Chimeric polypeptides and their use in bacterial decolonization

ABSTRACT

The present invention relates to assays, kits and oligonucleotides for the detection of  Pseudomonas aeruginosa  for a fast, sensitive and reliable detection of  Pseudomonas aeruginosa  in a species- and serotype-specific manner. In particular, the present invention provides an assay for the serotype-specific detection of  Pseudomonas aeruginosa , a kit for the serotype-specific detection of  Pseudomonas aeruginosa , as well as oligonucleotides useful in such assay or kit. The present invention further relates to the use of  Pseudomonas aeruginosa  serotype specific antibodies for serotype specific treatment of  Pseudomonas aeruginosa  infection in a patient detected for said specific  Pseudomonas aeruginosa  serotype with such an assay or kit.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 14/709,118, filed May 11, 2015, now allowed, which is adivisional of U.S. patent application Ser. No. 13/519,095, filed on Sep.17, 2012, now U.S. Pat. No. 9,057,059, which is a 35 U.S.C. § 371national phase application from, and claiming priority to, InternationalApplication No. PCT/EP2010/007941, filed Dec. 23, 2010, and publishedunder PCT Article 21(2) in English, which claims priority to EuropeanApplication No. EP 09015998.9, filed Dec. 23, 2009, all of whichapplications are incorporated by reference herein in their entireties.

TECHNICAL FIELD

The present invention relates to chimeric polypeptides comprising abacteriocin cell binding domain (CBD) and at least one enzymatic activedomain (EAD) having bacterial cell wall lytic activity, which are usefulin therapy and prophylaxis of pathogenic bacterial colonisation,including bacterial infections and bacterial diseases.

BACKGROUND OF THE INVENTION

The rapidly increasing number of antibiotic resistant bacteria is agrowing challenge for medicine and health care systems worldwide.Especially, the number of infections with methicillin-resistantStaphylococcus aureus (MRSA) increases dramatically in developedcountries. In the Netherlands, hospital acquired infections are managedsuccessfully by screening of patients entering hospital for MRSA carrierand consequent decolonisation of such patients. The established strategyof decolonisation of S. aureus from patients is eradication of S. aureusin nose using an antibiotic (for example, mupirocin) and decolonisationof skin using disinfectants. Problems of this decolonisation regimen are(i) extensive use of antibiotics, (ii) upcoming resistance of S. aureusagainst antibiotics (for example, against mupirocin), (iii) high costsdue to very long treatment time (normally 5-7 days), and (iv)opportunistic infections due to complete eradication of natural skinflora.

Considering these severe disadvantages, the community is seeking foralternative approaches for effective decolonisation within short timeand avoiding the use of antibiotics. Lysins, which arebacteriophage-induced lytic or hydrolytic enzymes responsible forbacterial host lysis, offer such advantages in general but they have tobe optimized for various aspects. Bacteriophage (phage) lysins have beendesignated using various names including lysins, phage-lysins,virolysins, and endolysins. Structurally, lysins are commonly found asmodular proteins with at least one domain that confers the enzymaticactivity for hydrolysing specific bonds in the murein or peptidoglycanlayer of the bacterial cell wall (enzymatic active domain—EAD), and onedomain that confers binding specificity to a carbohydrate epitope of thecell wall (cell binding domain—CBD). Thus, lysin family members (cellwall or peptidoglycan hydrolases) exhibit a modular design in which acatalytic domain is fused to a specificity or binding domain.

During the bacteriophage (phage) reproduction cycle, after assembly ofthe new phage particles, lysins (endolysins) are produced to destroy thebacterial cell wall. Endolysins can be divided in into five classesaccording to their cell wall lytic activities: (1) N-Acetylmuraminidases (lysozymes), (2) Endo-β-N-acetylglucosaminidases, (3) Lytictransglycosidases, (4) N-acetylmuramoyl-L-alanine amidases, and (5)Endopeptidases. While the aforementioned (1) to (3) all cleave withinthe sugar moiety of the peptidoglycan, (4) cleaves at the amid bondbetween the sugar backbone and the peptide linker and (5) cleaves withinthe peptid cross bridge.

Endolysins are described as having a narrow spectrum regarding theirtarget. In case of Gram-positive bacteria, endolysins act on cell wallsfrom inside as well as from outside, thus, making these molecules toantimicrobial drug candidates (Borysowski et al. 2006). Whilebacteriophage host ranges are largely restrictive, i.e. recognizing onlyone specific antigen on its bacterial host, phage lysins are lessrestrictive, recognizing a specific carbohydrate molecule common to theparticular species of host bacteria. Although the range of bacteriatargeted by lysins is less restrictive than the correspondingbacteriophage, lysins still maintain a degree of specificity, havingminimal effects on other bacteria including commensal organisms.

The use of endolysins to kill bacteria was disclosed for the first timeby Gasson in 1991 (GB 2 255 561). Further therapeutic and prophylacticapplications, including animal model systems, have been described byNelson et al. 2001. This work describes a topical application ofendolysins against group A streptococci and pneumococci in oral andnasopharyngeal treatment. In the field of staphylococcal treatment withbacteriophage derived lysins, Rashel et al. 2007 have shown thatendolysin from phage phiMR11 is able to eradicate MRSA in nares of miceand protects mice by intraperitoneal injection from septic death.Further regimes of treatment and pharmaceutical compositions to treatand prevent bacterial infections using phage derived lysins aredescribed in U.S. Pat. No. 5,997,862. However, in all so far publishedexamples using bacteriophage derived endolysins for the treatment ofbacterial infections, the amount of protein for an effective treatmentis very high. This is due the poor stability of the enzymes and due toinhibition of the activity in application relevant matrices.

In case of lysins against Staphylococcus bacteria, a number of wild-typeendolysins have been cloned and characterized. For example, protein 17from phage P68 is a staphylococcal endolysin, which is reported toexhibits antimicrobial activity against S. aureus isolates includingclinical isolates (Takác and Bläsi 2005). Various groups investigatedthe endolysin of S. aureus bacteriophage phi11 in antimicrobialapplications. Navarre et al. 1999 identified two enzymatic activitiesdomains (amidase and endopeptidase) in phi11 lysin and showed that amutant with deletion of the amidase domain is still active. Mutants ofphi11 (and phi12) endolysin have been characterized by differentactivity assays on S. aureus cell walls, heat inactivated bacteria andon bacterial biofilms (Sass and Bierbaum 2007). All these investigationshave in common that they are using artificial experimental conditionsfor functional characterization of the endolysins. Therefore, noevidences regarding efficacy on living cells under application-relevantconditions can be drawn from these publications.

Another staphylolytic enzyme is derived from bacteriophage phiK. Thisendolysin, called lysK, has been characterized in more detail by thegroups of David M. Donavan and R. Paul Ross (O'Flaherty et al. 2005; WO2008/001342; Becker et al. 2008; Horgan et al. 2009). They have beenable to show, that lysK has a broad bactericidal activity against livingStaphylococcus bacteria without discriminating between the differentgenera. LysK consists of one CBD and two EADs, a cysteine-histidineamino peptidase (CHAP) and an amidase domain. Expressing the individualEAD's, they were able to show that the CHAP domain alone is sufficientfor killing but not the amidase domain. A deletion mutant, withoutamidase domain (lysKΔ221-390), possesses the same killing activity asthe wild type protein. Determining MIC values for thetruncation/deletion constructs, only MIC values for the wild type LysKand the lysKΔ221-390 were measurable in TSB medium. The CHAP domainalone showed no measurable activity within such a complex matrix. Thedetermined MIC values are considerably high, 78 μg/ml and 63 μg/ml forwild type lysK and lysKΔ221-390, respectively. No chimeric lysin basedon lysK domains has been described so far.

All published data using wild type endolysins clearly show that thesemolecules are quite effective in killing bacteria in buffer solutions.The advantage of these molecules is the very fast onset time (minutes tohours), and the mode of action from outside without involvement ofmetabolic processes within the cell. As a matter of fact, for endolysinsinduction/acquisition of resistance has not been described inliterature. On the other hand, wild type endolysins tend to be quiteunstable at elevated temperatures and functionality is reduced incomplex compositions like culture media or biological fluids. Allpublished MIC values (minimal inhibitory concentration) or MBC values(minimal bactericidal concentration) are in the range>50 μg/ml. It canbe speculated that in many cases MIC values are not reported forexperimental reasons.

Enzymes with cell wall degrading properties similar to bacteriophagelysins (endolysins) can also be found in bacteria. Autolysins arebacteriolytic enzymes that digest the cell-wall peptidoglycan of thebacteria that produce them. Autolysins are involved in cell wallreconstruction during bacterial cell division. Although potentiallylethal, autolysins appear to be universal among bacteria that possesspeptidoglycan. “Autolysin” is the term used for lysins, which areproduced by bacteria and involved in cell division, while the term“lysin” or “endolysin” refers to lytic enzymes, which are involved inphage release, as described herein above. Bacteriocins are moleculesalso produced and secreted by microorganisms. They are antibacterialsubstances of a proteinaceous nature that are produced by differentbacterial species. A subclass of bacteriocins consists of enzymes(proteinaceous toxins) which are produced by bacteria to inhibit thegrowth of similar or closely related concurrence bacterial strain(s) intheir habitat. Many bacteria produce antimicrobial bacteriocin peptides.They also contain CBDs and EADs. Bacteriocins target prokaryotes but noteukaryotes, making them safe for human consumption.

The bacteriocin lysostaphin is naturally produced by Staphylococcussimulans to combat Staphylococcus aureus. It is highly effective invitro and capable of killing bacteria in complex media (Kumar J. 2008).Lysostaphin consists of one CBD and one glycyl-glycine endopeptidasedomain, which cleaves the characteristic penta-glycine cross bridge inS. aureus cell walls. This molecule has been tested in various animalmodels and exhibit good efficacy even in complex matrices (Kokai-Kun etal. 2007; Kusuma et al. 2007). The reported MIC values of lysostaphinare more than 1000-fold lower compared to lysK (<0.02 μg/ml). The majordisadvantage of lysostaphin is the occurrence of resistance in S.aureus. Two different genetic escape mechanisms have been described sofar: First, incorporation of serine into the penta-glycine bridge(DeHart et al. 1995). Secondly, shortening of the glycine bridge; gly3or gly2 (Ehlert et al. 1997; Strandén et al. 1997). It can be assumedthat such monogenic resistance marker will rapidly be selected underselection pressure.

Enzymatic active domains (EADs) can further be found in structuralbacteriophage proteins. They are part of the early infection machineryof the bacteriophage, locally hydrolyzing the cell wall prior to DNAinjection.

In order to deal with the fact of resistance development, groups startedto investigate the combination of different lysins. For example,synergistic effects between lysK and lysostaphin (Becker et al. 2008)have been described, resulting in reduced effective concentrations forkilling S. aureus. The drawback of this concept is, that in case ofoccurrence of resistance against one component (for example,lysostaphin), the concentration of the second component will not beeffective anymore. Furthermore, a composition with two active componentsis difficult to develop and expensive in production.

It is known that a combination of domains (CBD's and EAD's) fromdifferent source organisms is possible. However, the purpose of suchdomain exchange experiments was always to alter or broaden the hostspecificity of the lysins (Diaz et al. 1990; Croux et al. 1993; Donovanet al. 2006). So far, no systematic domain exchange experiments havebeen performed with endolysin-derived EAD's to obtain lytic moleculeswith improved properties with respect to efficacy, resistance potentialand stability.

There is one example in the literature to construct a highly stablechimeric lysin based on a lysostaphin CBD fused to a tail associatedmurein-degrading enzyme (TAME) domain (WO 2007/130655). This domain canbe considered as stable as it is a part of a bacteriophage structuralprotein. The disadvantage of such constructs is the very low specificactivity compared to endolysins. Therefore, more protein is required toreach effective concentrations. Furthermore, inhibition of the moleculesin complex matrices cannot be excluded because no characterization inthis regard has been provided.

There is an ongoing need for therapies and agents effective in thecontrol of bacterial contamination, colonization and infection. A majorproblem in medicine has been the development of drug resistant bacteriaas more antibiotics are used for a wide variety of illnesses and otherconditions. The over utilization of antibiotics has increased the numberof bacteria showing resistance. Furthermore, broadly reactiveantibiotics can affect normal flora and can cause antibiotic resistancein these organisms because of the frequency of drug use. The number ofpeople becoming hyper allogenic to antibiotics appears to be increasingbecause of antibiotic overutilization. Accordingly, there is acommercial need for new antibiotics (or bacterial killing substances),especially those that operate in new modalities or provide new means tokill pathogenic bacteria.

SUMMARY OF THE INVENTION

The use of lytic domains of a bacteriophage endolysin, a bacteriocin ora bacterial autolysin, specifically lytic domains of bacteriophagederived endolysins, for the treatment of bacterial infections is apromising alternative to overcome the increasing number of antibioticresistance in bacteria. As shown in principle by a number ofinvestigators, it is possible to kill bacteria in vitro and in animalmodels. Advantage of such lytic proteins is the fast onset of action andthe lower risk of resistance development against these enzymes. As ageneral drawback, all the studies so far have shown that relatively highconcentration of lysine is required for complete eradication of thetarget bacteria. The reason for the need of such high concentrations canbe explained with reduced activity of the molecules in complex matricesand their low stability at elevated temperature. Application relevantactivity data like (i) MIC values in bacterial growth media, (ii) MBCvalues in application relevant matrices (serum, growth media, mucinetc.), (iii) Log values of cfu reduction in relevant matrices (serum,growth media, mucin etc.), (iv) lysin activity in dependence ofbacterial growth phase, and (v) pH range of activity, have thereforebeen rarely published. A further disadvantage of current staphylococcallysins is that they tend to be rather unstable and often show poorsolubility.

The present invention successfully provides new chimeric polypeptidesagainst Gram-positive bacteria, Staphylococcus aureus, specificallyincluding MRSA, with substantially improved efficacy in relevantmatrices like culture media, mucin or serum. In addition, the chimericpolypeptides according to the present invention exhibit excellentthermal stability and good solubility. The activity of the chimericpolypeptides according to the present invention may not be dependent onthe bacterial growth phase. The chimeric polypeptides provided by thepresent invention are useful in the treatment and prophylaxis ofpathogenic bacterial colonisation, including bacterial infections andbacterial diseases, specifically pathogenic Gram-positive bacteriaincluding pathogenic Staphylococcus bacteria.

The present invention provides the following items:

[1] A chimeric polypeptide comprising a first portion and a secondportion joined by a linker, wherein

(a) said first portion comprises an amino acid sequence of a bacteriocincell binding domain (CBD); and

(b) said second portion comprises an amino acid sequence of at least oneenzymatic active domain (EAD) selected from

(i) the lytic domain of a bacteriophage lysin;

(ii) the lytic domain of a bacteriocin; and

(iii) the lytic domain of a bacterial autolysin.

[2] The chimeric polypeptide of item [1], wherein the lytic domain of(i) has at least 80%, preferably 90%, amino acid sequence identity withthe polypeptide of SEQ ID NO: 1.

[3] The chimeric polypeptide of item [1], wherein the lytic domain of(ii) has at least 80%, preferably 90%, amino acid sequence identity withthe polypeptide of SEQ ID NO: 2.

[4] The chimeric polypeptide of item [1], wherein the lytic domain of(iii) has at least 80%, preferably 90%, amino acid sequence identitywith the polypeptide of SEQ ID NO: 3.

[5] The chimeric polypeptide of any one of items [1] to [4], wherein theCBD has at least 80%, preferably 90%, amino acid sequence identity withthe polypeptide of SEQ ID NO: 4.

[6] The chimeric polypeptide of any one of items 1 to 5, having at least80%, preferably 90%, amino acid sequence identity with the polypeptideof SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 15, andhaving essentially the same biological activity as the correspondingpolypeptide of SEQ NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:15.

[7] The chimeric polypeptide of any one of items [1] to [6], having aMIC value ≤10 μg/ml, preferably ≤1 μg/ml, and more preferably ≤0.1μg/ml.

[8] The chimeric polypeptide of any one of items [1] to [6], having anMBC (99.99%) value of ≤0.5 μg/ml, preferably ≤0.05 μg/ml.

[9] The chimeric polypeptide of any one of items [1] to [6], having athermal stability (Tm) ≤45° C., preferably ≤50° C.

[10] The chimeric polypeptide of any one of items [1], [2], [5] and [6],wherein the bacteriophage lysin is a bacteriophage endolysin.

[11] The chimeric polypeptide of item [10], wherein the bacteriophageendolysin is lysK.

[12] The chimeric polypeptide of item [11], wherein the lytic domain isthe lytic CHAP domain of lysK.

[13] The chimeric polypeptide of any one of items [1], [3], [5] and [6],wherein the bacteriocin lytic domain is the lytic domain of lysostaphin.

[14] The chimeric polypeptide of any one of items [1] and [4] to [6],wherein the bacterial autolysin is LytN.

[15] The chimeric polypeptide of any one of items [1] to [14], whereinthe lytic domain exhibits the activity of an amidase, an endopeptidase,or a glycosidase.

[16] The chimeric polypeptide of item [15], wherein the glycosidase is amuramidase, a glucosaminidase, or a transglycosylase.

[17] The chimeric polypeptide of item [15], wherein the amidase is aN-acetylmuramyl-L-alanine amidase.

[18] The chimeric polypeptide of item [15], wherein the peptidase is aD-alanyl-glycyl-endopeptidase or a glycyl-glycyl-endopeptidase.

[19] The chimeric polypeptide of any one of items [1] to [18], whereinthe CBD is a lysostaphin CBD.

[20] The chimeric polypeptide of any one of items [1] to [19], whereinthe linker comprises at least one peptide bond.

[21] A nucleic acid molecule encoding the chimeric polypeptide of anyone of items [1] to [20].

[22] A composition comprising the chimeric polypeptide of any one ofitems [1] to [20].

[23] A formulation, preferably a topical formulation, comprising thechimeric polypeptide of any one of items [1] to [20].

[24] The formulation of item [23], which is in the form of abioadhesive, a medicated plaster, or a skin patch.

[25] The chimeric polypeptide of any one of items [1] to [20], thecomposition of item [22], or the formulation of item [23] or [24], foruse in prophylaxis or therapy.

[26] The chimeric polypeptide of any one of items [1] to [20], or thecomposition of item [22], for use in treating or preventing a bacterialdisease, a bacterial infection or bacterial colonization.

[27] Use of the chimeric polypeptide of any one of items [1] to [20], orthe composition of item [22], for the preparation of a medicament fortreating or preventing a bacterial disease, a bacterial infection orbacterial colonization.

[28] The chimeric polypeptide or composition of item [26], or the use ofitem [27], wherein the lytic activity

(a) decreases the occurrence or severity of a local or systemicbacterial disease or bacterial infection, or

(b) prevents or eliminates bacterial colonization.

[29] The chimeric polypeptide, composition or use of item [28], whereinthe bacterial disease, bacterial infection or bacterial colonization arecaused by Gram-positive bacteria.

[30] The chimeric polypeptide, composition or use of item [29], whereinthe Gram-positive bacteria is Staphylococcus, preferably Staphylococcusaureus, and more preferably methicillin-resistant Staphylococcus aureus(MRSA).

[31] The chimeric polypeptide or composition of any one of items [26]and [28] to [30], or the use of any one of items [26] to [30], whereinthe bacterial disease, bacterial infection or bacterial colonization isa bacterial disease, a bacterial infection or bacterial colonization ofthe skin or a mucous membrane, preferably the mucous membrane of theupper respiratory tract, more preferably the mucous membrane of thenasal cavity.

[32] The chimeric polypeptide, composition or use of item [31], furthercomprising a pharmaceutically acceptable carrier.

[33] The chimeric polypeptide, composition or use of item [32], whereinthe carrier is aqueous, preferably selected from the group consisting ofa cream, a gel, a lotion, and a paste.

The chimeric polypeptides of the present invention are targeted againstspecific pathogenic bacteria and these do not interfere with the normalbacterial flora. Also, chimeric polypeptides of the present inventionprimarily attack cell wall structures, which are not affected by plasmidvariation. The actions of the enzymatic active domains of the chimericpolypeptides of the present invention are fast and may not depend onbacterial growth. The chimeric polypeptides of the present invention canbe directed to the mucosal lining, where, in residence, they can killcolonizing bacteria, specifically Gram-positive bacteria, morespecifically Staphylococcus strains, still more specifically species andsub-species of Staphylococcus aureus, and most specifically MRSA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of treatment of a human skin with a chimericpolypeptide of the present invention (Example 9). “Disinfectant”represents the positive control. “Before treatment” represents thenegative control.

FIG. 2, comprising FIG. 2A and FIG. 2B, shows the results of treatmentof Staphylococcus aureus on LB-Agar plates with a chimeric polypeptideof the present invention (Example 10). FIG. 2A represents the controlexperiment. FIG. 2B represents the treated gel containing 10 μg PRF102.

FIG. 3 shows pH-dependent activity of PRF119 on a Staphylococcus aureusculture.

DETAILED DESCRIPTION OF THE INVENTION

Chimeric polypeptides provided by the present invention have killingactivity against two or more bacterial strains, preferably Gram-positivebacterial strains. In one aspect, the chimeric polypeptides exhibit alytic effect on multiple bacterial strains, including multipleGram-positive strains, preferably Staphylococcus strains, morepreferably Staphylococcus aureus strains. Specifically, the presentinvention provides chimeric polypeptides for the treatment andprevention of Staphylococcus aureus infections.

The foregoing has outlined the features of various embodiments in orderthat the detailed description that follows may be better understood.Additional features and advantages of various embodiments will bedescribed hereinafter which form the subject of the claims of theinvention.

In one aspect, the chimeric polypeptides of the present invention areuseful for application, in particular topical application, fordecolonisation of Gram-positive bacteria, specifically Staphylococcusaureus and methicillin-resistant S. aureus (MRSA), based on theenzymatic activity of the lytic domain(s) in combination with improvedactivity, stability and specificity of the chimeric polypeptides. It wassurprisingly found that a chimeric polypeptide according to the presentinvention shows improved lytic activity, improved host selectivity andstability compared to wild type endolysins. Specifically, an improvedactivity (expressed by MIC value) and improved stability was found for achimeric polypeptide, which comprises a first portion and a secondportion, wherein said first portion comprises an amino acid sequence ofthe CBD of lysostaphin, preferably the CBD of SEQ ID NO: 4, and whereinsaid second portion comprises an amino acid sequence of the CHAP domainof lysK, preferably the CHAP domain of SEQ ID NO: 1. Such a chimericpolypeptide has also proven to exhibit an improved host selectivity,preferably for Staphylococcus strains.

Likewise, an improved activity (expressed by MIC value) and improvedstability was found for a chimeric polypeptide, which comprises a firstportion and a second portion, wherein said first portion comprises anamino acid sequence of the CBD of lysostaphin, preferably the CBD of SEQID NO: 4, and wherein said second portion comprises an amino acidsequence of the CHAP domain of lysK, preferably the CHAP domain of SEQID NO: 1, and an amino acid sequence of the lytic domain of lysostaphin,preferably the lytic domain of SEQ ID NO: 2. Such a chimeric polypeptidehas also proven to exhibit an improved host selectivity, preferably forStaphylococcus strains.

In another aspect, the chimeric polypeptides of the present inventionposses MIC values lower than 10 μg/ml, preferably lower than 1 μg/ml,still more preferably lower than 0.3 μg/ml, and most preferably lowerthan 0.1 μg/ml.

In a still further aspect, the chimeric polypeptides of the presentinvention posses MBC (99.99%) values for 4 log reduction of livingbacterial cells lower than 10 μg/ml, preferably lower than 1 μg/ml, morepreferably lower than 0.5 μg/ml, still more preferably lower than 0.05μg/ml, and most preferably lower than 0.01 μg/ml. It is an object of thepresent invention that the MBC values for 4 log reduction of bacterialcells of the chimeric polypeptides provided herein is not significantlyinhibited in complex matrices like mucin and/or blood and/or serum.

In one embodiment of the present invention, at least one enzymaticactive domain (EAD) is used, which cleaves between highly conservedresidues within the peptid cross bridge of S. aureus cell walls.

In one aspect, the present invention provides chimeric polypeptides,which are optimized as they allow discriminating between S. aureus andother staphylococcal species.

In a further aspect, the chimeric polypeptides of the present inventionare highly active and posses improved stability.

In the present invention, the term “CBD” represents the abbreviation forcell binding domain, more specifically cell wall binding domain. Thus,the term “CBD” may also represent the abbreviation for cell wall bindingdomain. The terms “cell binding domain” and “cell wall binding domain”may be used interchangeably. The structural and functional definition ofa CBD according to the present invention is given elsewhere in thedescription.

In the present invention, “EAD” represents the abbreviation forenzymatic active domain. The structural and functional definition of anEAD according to the present invention is given elsewhere in thedescription.

A “chimeric polypeptide” according to the present invention is acombination of a first portion and a second portion, wherein the firstportion comprises an amino acid sequence of a bacteriocin cell bindingdomain (CBD) and the second portion comprises at least one enzymaticactive domain (EAD), wherein the domains stem from a different source ordifferent origin. Specifically, a “chimeric polypeptide” according tothe present invention is a combination of a first portion and a secondportion, wherein the first portion comprises an amino acid sequence of abacteriocin cell binding domain (CBD) and the second portion comprisesat least one enzymatic active domain (EAD), wherein the domains stemfrom a different source organism, or source enzyme. In other words, thedomains stem from a different origin of organism or different origin ofenzyme. In this context, the terms “protein” and “peptide” may be usedinterchangeably with the term “enzyme”. In other words, a “chimericpolypeptide” of the present invention is a polypeptide, which comprisesheterologous domains.

In the present invention, the chimeric polypeptide comprises a first anda second portion, wherein the first portion generally comprises an aminoacid sequence of a bacteriocin CBD. Bacteriocins are molecules producedby microorganisms. Thus, if the second portion of the chimericpolypeptide comprises an amino acid sequence of the lytic domain of abacteriophage lysin as the EAD, the chimeric polypeptide is chimeric dueto the fact that the CBD stems from a microorganism while the EAD stemsfrom a bacteriophage.

In one aspect of the present invention, a “chimeric polypeptide”according to the present invention is a combination of a first portionand a second portion, wherein the first portion comprises an amino acidsequence of a bacteriocin cell binding domain (CBD) and the secondportion comprises at least one enzymatic active domain (EAD), whereinthe EAD is the lytic domain of a bacteriophage lysin. Preferably, theEAD is the lytic domain of a bacteriophage endolysin. More preferably,the EAD is the lytic domain of a bacteriophage endolysin, wherein thebacteriophage is from a Gram-positive bacterium. Still more preferably,the EAD is the lytic domain of a bacteriophage endolysin, wherein thebacteriophage is from a species or sub-species of Staphylococcus. In amore preferred embodiment, the EAD is the lytic domain of lysK,specifically the CHAP domain of lysK. Most preferably, the EAD comprisesthe amino acid sequence of SEQ ID NO: 1.

In another aspect, a “chimeric polypeptide” according to the presentinvention is a combination of a first portion and a second portion,wherein the first portion and the second each comprise an amino acidsequence of domains, CBD and EAD(s), wherein the CBD and EAD(s) are fromdifferent bacteriophages. More preferably, the domains are fromdifferent bacteriophages infecting Gram-positive bacteria.

In one aspect of the present invention, a “chimeric polypeptide”according to the present invention is a combination of a first portionand a second portion, wherein the first portion comprises an amino acidsequence of a bacteriocin cell binding domain (CBD) and the secondportion comprises at least one enzymatic active domain (EAD), whereinthe EAD is the lytic domain of a bacteriocin. Preferably, the EAD is thelytic domain of bacteriocin from a Gram-positive bacterium. Morepreferably, the EAD is the lytic domain of a bacteriocin from a speciesor sub-species of Staphylococcus, specifically from S. simulans. Stillmore preferably, the EAD is the lytic domain of lysostaphin. Mostpreferably, the EAD comprises the amino acid sequence of SEQ ID NO: 2.

In one aspect of the present invention, a “chimeric polypeptide”according to the present invention is a combination of a first portionand a second portion, wherein the first portion comprises an amino acidsequence of a bacteriocin cell binding domain (CBD) and the secondportion comprises at least one enzymatic active domain (EAD), whereinthe EAD is the lytic domain of a bacterial autolysin. If the EAD is thelytic domain of a bacterial autolysin, the chimeric polypeptide ischimeric due to the fact that the CBD stems from a bacteriocin while theEAD stems from a bacterial autolysin. The person skilled in the art isfully aware of proteins, which are classified as being bacteriocins andthose, which are classified as being bacterial autolysins. In apreferred embodiment, a “chimeric polypeptide” according to the presentinvention is a combination of a first portion and a second portion,wherein the first portion comprises an amino acid sequence of abacteriocin cell binding domain (CBD) and the second portion comprisesat least one enzymatic active domain (EAD), wherein the EAD is the lyticdomain of an autolysin from a Gram-positive bacterium. More preferably,the EAD is the lytic domain of an autolysin from a species orsub-species of Staphylococcus. Still more preferably, the EAD is thelytic domain of lytN or lytM, specifically the CHAP domain of lytN orlytM. Most preferably, the EAD comprises the amino acid sequence of SEQID NO: 3.

As defined herein, the first portion of the chimeric polypeptide of thepresent invention is defined to comprise an amino acid sequence of abacteriocin cell wall-binding domain (CBD). Furthermore, as definedherein, the second portion of the chimeric polypeptide of the presentinvention is defined to comprise an amino acid sequence of at least oneenzymatic active domain (EAD) selected from the lytic domain of abacteriophage lysin, the lytic domain of a bacteriocin, and the lyticdomain of a bacterial autolysin. Thus, by way of definition, one mayconsider that the chimeric polypeptide of the present invention maycomprise the cell binding domain of a bacteriocin and the lytic domainof a bacteriocin. Lysostaphin is a bacteriocin, which comprises a cellbinding domain and a lytic domain in form of an endopeptidase. However,lysostaphin is excluded from the definition of a chimeric polypeptide ofthe present invention since by way of definition a chimeric polypeptideof the present invention comprises a first portion and a second portion,wherein the first portion comprises an amino acid sequence of abacteriocin cell binding domain (CBD) and the second portion comprisesat least one enzymatic active domain (EAD), wherein the domains stemfrom a different source organism or source enzyme. Thus, in the presentinvention, it is excluded that the CBD and the EAD both stem from thebacteriocin lysostaphin, specifically from the lysostaphin naturallyproduced by Staphylococcus simulans. At least by way of defining thepolypeptides of the present as being “chimeric”, i.e. comprising“heterologous” domains as explained herein above, lysostaphin isexcluded from this definition since lysostaphin is not composed ofheterologous domains.

In the present invention, the source or origin of the domains comprisedby the first and second portion of the chimeric polypeptide of thepresent invention is different. This does not mean that while the firstportion comprises by way of definition a bacteriocin CBD, the secondportion may not comprise at least one enzymatic active domain (EAD),which is a lytic domain of a bacteriocin, although lysostaphin isexcluded as a chimeric polypeptide of the present invention. That is,the CBD and the EAD of the chimeric polypeptide of the present inventionmay not find their origin in the same bacteriocin, but may find theirorigin in different bacteriocins. Bacteriocins are different from eachother when they stem from a different source or origin of organism.Bacteriocins are also different from each other when they do notrecognize members of the same, but of closely related or differentspecies by binding receptor sites on sensitive, or susceptible,organisms. Furthermore, a chimeric polypeptide of the present inventionmay comprise a CBD and an EAD from the same source or origin oforganism, provided that the chimeric polypeptide comprises at least onefurther EAD from a different source or origin of organism or from adifferent source or origin of enzyme.

A chimeric polypeptide according to the present invention may comprisemore than one enzymatic active domain and, thus, can act on differentmolecules, and hence has the potential to treat two or more differentbacterial infections at the same time. Likewise, a chimeric polypeptideaccording to the present invention may also be used to treat a bacterialinfection by cleaving the cell wall in more than one location.

In one embodiment, a “chimeric polypeptide” according to the presentinvention is a combination of a first portion and a second portion,wherein the first portion comprises an amino acid sequence of abacteriocin cell binding domain (CBD) and the second portion comprisesat least one enzymatic active domain (EAD), wherein the domains are fromdifferent pathogenic bacterial species or different pathogenic bacterialsub-species, preferably from different pathogenic Gram-positivebacterial species or different pathogenic Gram-positive bacterialsub-species, and more preferably from different pathogenicStaphylococcus species or different pathogenic Staphylococcussub-species.

A pathogenic bacterial species or sub-species is defined by thesimilarities found among its members. Properties such as biochemicalreactions, chemical composition, cellular structures, geneticcharacteristics, and immunological features are used in defining apathogenic bacterial species or sub-species and thus differentiatingdifferent pathogenic bacterial species and sub-species.

In another embodiment, a “chimeric polypeptide” according to the presentinvention is a combination of a first portion and a second portion,wherein the first portion comprises an amino acid sequence of abacteriocin cell binding domain (CBD) and the second portion comprisesat least one enzymatic active domain (EAD), wherein the at least one EADstems from a bacteriophage.

The chimeric polypeptide of the present invention comprises a firstportion and a second portion joined by a linker, wherein said firstportion comprises an amino acid sequence of a bacteriocin cell bindingdomain (CBD). In a preferred embodiment, the bacteriocin CBD of thepresent invention is a CBD produced by a Gram-positive bacterium. Morepreferably, the bacteriocin CBD of the present invention is aStaphylococcus bacteriocin CBD. In a more preferred embodiment of thepresent invention, the bacteriocin CBD is the CBD of lysostaphin. Thebacteriocin lysostaphin is naturally produced by Staphylococcussimulans. Most preferably, in the present invention the bacteriocin CBDcomprises the amino acid sequence of SEQ ID NO: 4.

A bacteriocin CBD according to the present invention is supposed toencompass herein all those bacteriocin protein domains, which are partof bacteriocin proteins binding to a target bacterium, specifically tothe cell wall of a target bacterium. The cell binding domain or cellwall binding domain according to the present invention is that part of abacteriocin cell binding protein or bacteriocin cell wall bindingprotein, which is necessary and sufficient for the bacterial cellbinding ability, specifically the cell wall binding ability.

As described above, bacteriocin CBDs of the present invention aredefined as being derived from proteins or enzymes of bacteriocin origin,which are capable of specific binding to bacteria. In this context,“derived from” refers to those CBDs, which maintain their bindingability, but have no or no significant hydrolytic activity. No or nosignificant hydrolytic activity in this context is intended to describethe situation whereby the hydrolytic activity is not sufficient toprevent the application of a bacteriocin CBD to bind to a cell, morespecifically to a cell wall. A bacteriocin CBD according to the presentinvention is supposed to be a protein, which does not have anyhydrolytic activity itself. This also applies to fragments and variantsof a bacteriocin CBD according to the present invention, which aredescribed herein and which are also encompassed by the presentinvention.

A bacteriocin CBD according to the present invention binds to bacterialcells, specifically to cell walls of target bacteria, more specificallyto cell wall components coded by the target cell DNA, and still morespecifically to cell wall components coded by the target cell DNA, whichare non-covalently associated with the cell wall of a target cell.

The gene sequences coding for the bacteriocin CBDs according to thepresent invention can be derived from the corresponding geneticinformation of the cells, which code for the cell wall bindingdomains/proteins.

Bacteriophage lysins fall into three categories, glycosidases, amidases,and endopeptidases, depending on the type of chemical bond they cleavewithin the peptidoglycan. Glycosidases can be further subdivided intothe muramidases, glucosaminidases, and transglycosylases. In the presentinvention, bacteriophage lysins provide at least one of the followingenzymatic activities against a peptidoglycan substrate: muramidases,glucosaminidases, N-acetylmuramyl-L-alanine amidase and endopeptidases.

Bacteriophages are not only known to encode and produce lysins, but alsoso-called tail associated muralytic enzymes (TAMEs), which are likewisecapable of hydrolysing bacterial cell walls. While lysins are producedin the final stage of the phage-life cycle to facilitate the release ofprogeny phage from the host bacterium, TAMEs are, in contrast, producedduring the first stage of the process of infection of a host cell. Thefirst stage of the phage infection process comprises the steps ofadsorption to and penetration of the host cell, which is mediated using,inter alia, the TAME. Many but not all phages have tails attached to thephage head.

Bacteriophage lysins are structurally composed of two domains, anenzymatic active lytic domain and a cell binding domain. In the presentinvention, the second portion of the chimeric polypeptide may comprisean amino acid sequence of the lytic domain of a bacteriophage lysine.Excluded from the present invention are bacteriophage tail associatedmuralytic enzymes (TAMEs). Thus, while the second portion of thechimeric polypeptide of the present invention may comprise an amino acidsequence of the lytic domain of a bacteriophage lysine (endolysin), itis excluded that the lytic domain of a bacteriophage lysin according tothe present invention may be a so-called bacteriophage tail associatedmuralytic enzyme (TAME) as described herein above. In other terms,excluded from the present invention are tail portions of bacteriophagesor so-called bacteriophage tail associated muralytic enzymes (TAMEs)exhibiting the activity of hydrolysing bacterial cell walls.

In one aspect of the present invention, a Gram-positive bacterium ispreferably a pathogenic Gram-positive bacterium. More preferably, in thepresent invention a Gram-positive bacterium is a pathogenicStaphylococcus bacterium. Still more preferably, in the presentinvention a pathogenic Staphylococcus bacterium is a pathogenic speciesor sub-species of Staphylococcus. In one aspect of the presentinvention, a pathogenic Staphylococcus bacterium is preferably S. aureusor MRSA. In another aspect of the present invention, a pathogenicbacterium belonging to the genus Staphylococcus is preferably S.epidermidis or S. haemolyticus. In still another aspect of the presentinvention, a pathogenic bacterium belonging to the genus Staphylococcusis preferably S. simulans or S. saprophyticus. In a further aspect ofthe present invention, a pathogenic bacterium belonging to the genusStaphylococcus is preferably S. hyicus or S. warneri. In one aspect, apathogenic bacterium belonging to the genus Staphylococcus is S.xylosus.

The above definition applies to all aspects of the present invention,i.e., including the application of a chimeric polypeptide according tothe present invention in therapy or prophylaxis as well as thedefinition of the lytic domain(s) of the EAD(s) and the CBD comprised bythe chimeric polypeptide. For example, if the EAD is the lytic domain ofa bacteriophage endolysin, wherein the bacteriophage is from aGram-positive bacterium, the Gram-positive bacterium is preferably apathogenic Gram-positive bacterium. More preferably, the EAD is thelytic domain of a bacteriophage endolysin, wherein the bacteriophage isfrom a pathogenic Staphylococcus bacterium. Still more preferably, theEAD is the lytic domain of a bacteriophage endolysin, wherein thebacteriophage is from a pathogenic species or sub-species ofStaphylococcus, specifically from S. aureus. Likewise, if the EAD is thelytic domain of a bacteriocin, wherein the bacteriocin is from aGram-positive bacterium, the Gram-positive bacterium is preferably apathogenic Gram-positive bacterium. More preferably, the EAD is thelytic domain of a bacteriocin from a pathogenic Staphylococcusbacterium. Still more preferably, the EAD is the lytic domain of abacteriocin from a pathogenic species or sub-species of Staphylococcus,specifically from S. simulans. Likewise, if the EAD is the lytic domainof a bacterial autolysin from a Gram-positive bacterium, theGram-positive bacterium is preferably a pathogenic Gram-positivebacterium. More preferably, the EAD is the lytic domain of a bacterialautolysin from a pathogenic Staphylococcus bacterium. Still morepreferably, the EAD is the lytic domain of a bacterial autolysin from apathogenic species or sub-species of Staphylococcus, specifically fromS. aureus. Likewise, if the bacteriocin CBD of chimeric polypeptide ofthe present invention is the bacteriocin CBD of a Gram-positivebacterium, the Gram-positive bacterium is preferably a pathogenicGram-positive bacterium. More preferably, the bacteriocin CBD is from apathogenic Staphylococcus bacterium. Still more preferably, thebacteriocin CBD is from a pathogenic species or sub-species ofStaphylococcus, specifically from S. simulans.

In one preferred embodiment of the present invention the chimericpolypeptide comprises a first portion and a second portion joined by alinker, wherein said first portion comprises an amino acid sequence ofthe CBD of lysostaphin, preferably the CBD of SEQ ID NO: 4, and whereinsaid second portion comprises an amino acid sequence of the CHAP domainof lysK, preferably the CHAP domain of SEQ ID NO: 1 (PRF119 and PRF133).Herein, the CBD of lysostaphin is fused with its N-terminus to theC-terminus of the CHAP domain of lysK. In a preferred embodiment, such achimeric polypeptide has a MIC value ≤10 μg/ml, preferably ≤1 μg/ml, andmore preferably ≤0.1 μg/ml. Furthermore, such a chimeric polypeptide haspreferably an MBC (99.99%) value of ≤0.5 μg/ml, preferably ≤0.05 μg/ml.

In a further preferred embodiment of the present invention the chimericpolypeptide comprises a first portion and a second portion joined by alinker, wherein said first portion comprises an amino acid sequence ofthe CBD of lysostaphin, preferably the CBD of SEQ ID NO: 4, and whereinsaid second portion comprises an amino acid sequence of the CHAP domainof lysK, preferably the CHAP domain of SEQ ID NO: 1, and an amino acidsequence of the lytic domain of lysostaphin, preferably the lytic domainof SEQ ID NO: 2 (PRF115). Herein, lysostaphin is fused with itsN-terminus to the C-terminus of the CHAP domain of lysK. In a preferredembodiment, such a chimeric polypeptide has a MIC value ≤10 μg/ml,preferably ≤1 μg/ml, and more preferably ≤0.1 μg/ml. Furthermore, such achimeric polypeptide has preferably an MBC (99.99%) value of ≤0.5 μg/ml,preferably ≤0.05 μg/ml.

In a still further preferred embodiment of the present invention thechimeric polypeptide comprises a first portion and a second portionjoined by a linker, wherein said first portion comprises an amino acidsequence of the CBD of lysostaphin, preferably the CBD of SEQ ID NO: 4,and wherein said second portion comprises an amino acid sequence of theCHAP domain of lytN, preferably the CHAP domain of SEQ ID NO: 3(PRF102). Herein, the CBD of lysostaphin is fused with its N-terminus tothe C-terminus of the CHAP domain of lytN. In a preferred embodiment,such a chimeric polypeptide has a MIC value ≤10 μg/ml, preferably ≤1μg/ml, and more preferably ≤0.1 μg/ml. Furthermore, such a chimericpolypeptide has preferably an MBC (99.99%) value of 50.5 μg/ml,preferably ≤0.05 μg/ml.

The lytic domain of lysostaphin has a specific lytic action againstStaphylococcus. In particular, the lytic domain of lysostaphin hasglycyl-glycine endopeptidase activity. Accordingly, in one aspect of thepresent invention the lytic domain of the chimeric polypeptide of theinvention is the lytic domain of lysostaphin, which has glycyl-glycineendopeptidase activity. In a preferred embodiment, the lytic domain ofthe chimeric polypeptide of the present invention is the lytic domain oflysostaphin and thus the chimeric polypeptide is used in prophylaxis ortherapy of staphylococcal infections and/or staphylococcalcolonisations.

The enzymatic activity of an enzymatic active domain (EAD) of a chimericpolypeptide of the present invention refers to a polypeptide having theactivity of lysing a bacterium whose cell wall contains peptidoglycan.Preferably, the bacterium having a cell wall containing peptidoglycan isa Gram-positive bacterium. The nature of peptidoglycan is known to theperson skilled in the art as a polymer of amino sugars cross-linked byshort peptides which forms a covalent matrix that surrounds thecytoplasmic membrane and constitutes the major skeletal component of thecell wall.

The lytic domains of the present invention can be isolated from natureor can be produced by recombinant or synthetic means. The term “lyticdomain” specifically encompasses naturally occurring forms (e.g.,alternatively spliced or modified forms) and naturally-occurringvariants of the enzyme. In one example, the native sequence enzyme is amature or full-length polypeptide that is genetically coded for by agene from a bacteriophage specific for pathogenic staphylococci,preferably methicillin-resistant Staphylococcus aureus (MRSA).

A “phage” or “bacteriophage”, as used herein, relates to the well-knowncategory of viruses that infect bacteria. Phages include DNA or RNAsequences encapsidated in a protein envelope or coat (“capsid”).

The term “CHAP” used in the context of the present invention is known tothe person skilled in the art as cysteine, histidine-dependentamidohydrolases/peptidases.

In the present invention, the term “bacterium” preferably describes a“target bacterium”, and refers to a bacterium that is bound by achimeric polypeptide of the present invention and and/or whose growth,survival, or replication is inhibited by the enzymatic activity of theenzymatic active domain (EAD) of the second portion of the chimericpolypeptide according to the present invention. The inhibition ofbacterial growth refers to the slowing or stopping of the rate of abacteria cell's division or cessation of bacterial cell division, or todeath of bacteria. The term “target bacterium” specifically includesGram-positive target bacteria.

“MIC” refers to minimum inhibitory concentration. The MIC value isdefined as the lowest concentration of a chimeric polypeptide of thepresent invention that prevented visible growth of test bacteria. MICassays were determined by the broth dilution method in a modification ofstandards of the NCCLS (2003; Methods for dilution antimicriobialsusceptibility test for bacteria that grow aerobically; approvedstandard M7-A6). The concentration of chimeric polypeptides used rangedfrom 200 μg/ml to 0.00019 μg/ml. Twofold dilutions were performed inBrain Heart Infusion Broth (BHI) supplemented with 0.1% bovine serumalbumin (BSA) in a 96-well microtiter plate. Each well was inoculatedwith 1×10⁵ CFU/ml Staphylococcus diluted from an overnight culture grownin BHI. As a control growth without protein was included. The microtiterplate was incubated at 30° C. for 24 hours. Values were determined bymeasuring the absorbance at 620 nm in a microplate reader.

“MBC” refers to minimum bactericidal concentration. MBCs for chimericpolypeptides were determined by a modification of the NCCLS standards(1999; Methods for determining bactericidal activity on antimicrobialagents; approved guideline Vol. 19). The concentration of chimericpolypeptides used ranged from 50 μg/ml to 0.00005 μg/ml. Tenfolddilutions were performed in 20 mM Tris/HCl pH 8, 60 mM NaCl, 2 mM CaCl₂supplemented with 1% bovine serum albumin (BSA) in reaction tubes.Staphylococcus from an overnight culture in BHI were diluted to a finalinoculum of 1×10⁵ CFU/ml in each tube. A tube containing buffer and BSAbut no protein was included as a control. The dilution tubes wereincubated at 30° C. for 1 hour. A volume of each sample (100 μl) wasplated on LB-Agar plates. The MBC value was defined as the dose ofchimeric polypeptide which led to a 3 log or greater drop from thestarting bacterial concentration (99.9% killing of the initialinoculum).

“Polypeptide” refers to a molecule comprised of amino acids whichcorrespond to polypeptides encoded by a polynucleotide sequence which isnaturally occurring. The polypeptide may include conservativesubstitutions where the naturally occurring amino acid is replaced byone having similar properties, where such conservative substitutions donot alter the function of the polypeptide (see, for example, Lewin“Genes V” Oxford University Press Chapter 1, pp. 9-13 1994). The terms“polypeptide”, “peptide”, and “protein” are typically usedinterchangeably herein to refer to a polymer of amino acid residues.Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission.

The present invention also relates to fragments and variants of thechimeric polypeptides provided herein. Specifically, provided herein arefragments and variants of the CBDs and EADs according to the presentinvention. In one aspect, provided herein is a variant of the lyticdomain of a bacteriophage lysine or endolysin described herein, whichhas at least 80%, preferably 90%, more preferably 95%, amino acidsequence identity with the polypeptide of SEQ ID NO: 1. In variousaspect, provided herein is a variant of the lytic domain of abacteriophage lysine or endolysin described herein, which has at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, preferably at least 91%,92%, 93%, or 94%, and more preferably 96%, 97%, 98%, or 99%, amino acidsequence identity with the polypeptide of SEQ ID NO: 1. In anotheraspect, provided herein is a variant of the lytic domain of abacteriocin described herein, which has at least 80%, preferably atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%, amino acid sequence identity with thepolypeptide of SEQ ID NO: 2. In still another aspect, provided herein isa variant of the lytic domain of a bacterial autolysin described herein,which has at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,amino acid sequence identity with the polypeptide of SEQ ID NO: 3. Inyet a further aspect, provided herein is a variant of the bacteriocinCBD described herein, which has at least 80%, preferably at least 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%, amino acid sequence identity with the polypeptideof SEQ ID NO: 4. Preferably, these variants have essentially the samebiological activity as the corresponding polypeptides of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

Also provided herein are variants of the chimeric polypeptides of SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 15, respectively,wherein each of the variants has at least 80%, preferably at least 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%, amino acid sequence identity with thecorresponding polypeptide of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,and SEQ ID NO: 15, respectively. In a preferred embodiment, suchvariants have a MIC value ≤10 μg/ml, preferably ≤1 μg/ml, and morepreferably ≤0.1 μg/ml. Furthermore, such variants have preferably an MBC(99.99%) value of ≤0.5 μg/ml, preferably ≤0.05 μg/ml.

“Percent (%) polypeptide sequence identity” with respect to thepolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific reference amino acid sequence, afteraligning the sequences in the same reading frame and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, such as using publicly available computer softwaresuch as blast software.

The invention provides variants of the above mentioned chimericpolypeptides, which increase stability and/or activity thereof.

The present invention provides fragments of the chimeric polypeptides ofthe invention as well as fragments of the CBDs and EADs of the presentinvention, which still exhibit the biological activity of a chimericpolypeptide or CBD and EAD, respectively, according to the presentinvention. As used herein, a “fragment” is a polypeptide variant havingan amino acid sequence that entirely is the same as part but not all ofthe amino acid sequence of the reference polypeptide. A fragment may be“free-standing” or comprised as a single continuous region within alarger polypeptide of which they form a part or region. In one aspect,provided herein is a fragment of the amino acid sequence of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, and SEQ ID NO: 15, respectively, wherein one or moreamino acid residues are deleted in the respective amino acid sequence,as long as the fragment exhibits the activity of hydrolysing a bacterialcell wall, preferably a Gram-positive bacterial cell wall. On anotheraspect, provided herein is a fragment of the amino acid sequence of SEQID NO: 4, wherein one or more amino acid residues are deleted in theamino acid sequence of SEQ ID NO: 4, as long as the fragment exhibitsthe activity of binding to a bacterial cell wall, preferably to the cellwall of a Gram-positive bacterium. In yet another aspect of the presentinvention, provided herein is a fragment of the amino acid sequence ofSEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 15,respectively, wherein one or more amino acid residues are deleted in theamino acid sequence of the respective polypeptide, as long as thefragment exhibits the activity of inhibition of bacterial growth,including the slowing or stopping of the rate of a bacteria cell'sdivision or cessation of bacterial cell division, or to death ofbacteria (killing colonizing bacteria). Preferably, such bacteria arepathogenic bacteria, more preferably pathogenic Gram-positive bacteria.

Biologically active portions of a protein or peptide fragment of theembodiments, as described herein, include polypeptides comprising aminoacid sequences sufficiently identical to or derived from the amino acidsequence of the EADs and CBDs according to the present invention, whichinclude fewer amino acids than the full length protein and exhibit thesame activity of the corresponding full length protein. A biologicallyactive portion of a protein or protein fragment of the present inventioncan be a polypeptide which is, for example, 5, 10, 15 or more aminoacids less in length than the reference polypeptide sequence.Degradation forms of the polypeptides of this embodiment in a host cellare also provided in some embodiments.

The present invention provides nucleic acids encoding the chimericpolypeptides of the present invention. Furthermore, provided herein arenucleic acids encoding the CBDs and EADs according to the presentinvention. Also provided are vectors carrying such nucleic acids andhost cells transformed or transfected with such vectors.

As used herein, a “nucleic acid” typically refers to deoxyribonucleotideor ribonucleotides polymers (pure or mixed) in single- ordouble-stranded form. The term may encompass nucleic acids containingnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding, structural, or functional properties as the referencenucleic acid and which are metabolized in a manner similar to thereference nucleotides. Non-limiting examples of such analogs include,without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,and peptide-nucleic acids (PNAs). The term nucleic acid may, in somecontexts, be used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

Embodiments of the disclosure include vectors that comprise apolynucleotide or polynucleotides encoding one of the polypeptidesequences described herein, or variants or fragments thereof. Otherexamples concern host cells that are genetically engineered with vectorsof the disclosure and the production of polypeptides of the disclosureby recombinant techniques. Cell-free translation systems can also beemployed to produce such proteins using RNAs derived from the DNAconstructs of the disclosure.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe disclosure. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals.Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci, E. coli, Streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the disclosure. Such. vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques. For secretion of the translated protein into thelumen of the endoplasmic reticulum, into the periplasmic space or intothe extracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the disclosure can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. High performance liquidchromatography is also employed for purification. Well-known techniquesfor refolding protein may be employed to regenerate active conformationwhen the polypeptide is denatured during isolation and or purification.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of the invention.

The fragments and variants of the polypeptides of the present inventiondescribed herein above include proteins or peptides and peptidefragments that are chemically synthesized or prepared by recombinant DNAtechniques, or both. Such fragments and variants may interchangeably bedescribed as modified or altered forms of the proteins or peptides ofthe present invention. In the present invention, peptide variants alsoinclude fragments of a polypeptide. When the protein or peptide isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals that are involved in thesynthesis of the protein. Such polypeptide variants include, forinstance, polypeptides wherein one or more amino acid residues areadded, or deleted at the N or C terminus of the sequence of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, or SEQ ID NO: 15. In an embodiment one or more aminoacids are substituted, deleted, and/or added to any position(s) in thesequence, or sequence portion thereof.

Variants that are fragments of the polypeptides of the disclosure may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of embodimentsof the disclosure.

Peptide fragments of the present invention may be prepared by any of anumber of conventional techniques. Desired peptide fragments may bechemically synthesized. An alternative approach involves generatinglytic enzyme fragments by enzymatic digestion, e.g., by treating theprotein with an enzyme known to cleave proteins at sites defined byparticular amino acid residues, or by digesting the DNA with suitablerestriction enzymes and isolating the desired fragment. Yet anothersuitable technique involves isolating and amplifying a DNA fragmentencoding a desired polypeptide fragment, by polymerase chain reaction(PCR). Oligonucleotides that define the desired termini of the DNAfragment are employed at the 5′ and 3′ primers in the PCR. Preferably,EAD or CBD polypeptide fragments of the present invention share the samebiological activity with the reference EAD or CBD polypeptide disclosedherein in the sequence listing.

The chimeric polypeptide according to the present invention comprises alinker sequence. In one embodiment, “linker sequence” refers to an aminoacid sequence that joins the two portions of the chimeric polypeptide,or fragments or variants thereof. In general, as used herein, a linkeris an amino acid sequence that covalently links the polypeptides to forma fusion polypeptide. The linker comprises at least one peptide bond. Asappreciated by one of skill in the art, the linker can compriseadditional amino acids, such as glycine and other small neutral aminoacids.

The present invention also relates to the use of chimeric polypeptidesprovided by the present invention for the reduction of certain bacterialpopulations, including methods and compositions for the treatment ofvarious bacterial infections. Thus, the present invention also relatesto compositions and formulations comprising chimeric polypeptidesaccording to the present invention and the use of such compositions inprophylaxis or therapy of bacterial diseases, bacterial infections orbacterial colonisations. In one aspect, a composition or formulation ofthe present invention is a decontamination composition ordecontamination formulation. In another aspect, a composition orformulation of the present invention is a decolonisation composition ordecolonisation formulation. In yet another aspect, a composition orformulation of the present invention is a disinfectant.

In one aspect of the present invention, the chimeric polypeptides areapplied in a method for the treatment or prophylaxis of Staphylococcusinfections in a subject, in particular for the treatment or prophylaxisof infections by S. aureus, S. aureus (MRSA), S. epidermidis, S.haemolyticus, S. simulans, S. saprophyticus, S. chromogenes, S. hyicus,S. warneri and/or S. xylosus. The subject may be a human subject or ananimal, in particular animals used in livestock farming and/or dairyfarming such as cattle and pigs. The method of treatment encompasses theapplication of the chimeric polypeptide of the present invention to thesite of infection or site to be prophylactically treated againstinfection in a sufficient amount.

In particular, the method of treatment may be for the treatment orprophylaxis of infections, in particular by Staphylococcus aureus or S.aureus (MRSA), of the skin, of soft tissues, of bacteremia and/orendocarditis.

Furthermore, a chimeric polypeptide of the present invention may be usedprophylactically as sanitizing agent, in particular before or aftersurgery, or for example during hemodialysis. Similarly, prematureinfants and immunocompromised persons, or those subjects with need forprosthetic devices can be treated with a chimeric polypeptide of thepresent invention, either prophylactically or during acute infection. Inthe same context, nosocomial infections by Staphylococcus, in particularby S. aureus or S. aureus (MRSA), may be treated prophylactically orduring acute phase with a chimeric polypeptide of the present invention.In this embodiment, a chimeric polypeptide of the present invention maybe used as a disinfectant also in combination with other ingredientsuseful in a disinfecting solution like detergents, tensids, solvents,antibiotics, lanthibiotics, or bacteriocins.

In a particular embodiment, a chimeric polypeptide of the presentinvention is used for medical treatment, if the infection to be treated(or prevented) is caused by multiresistant Staphylococcus strains, inparticular by strains resistant against vancomycin, linezolid ordaptomycin.

A composition as disclosed herein may comprise more than one chimericpolypeptide according to the present invention and/or may comprise oneor more additional agents. Non-limiting examples of an additional agentinclude an enzyme, an antibiotic, an anti-fungal agent, a bactericide,an analgesic, and an anti-inflammatory agent.

A composition or formulation according to the present inventionpreferably comprises a carrier suitable for delivering the chimericpolypeptide to the site of the bacterial disease, bacterial infection orbacterial colonisation. The compositions and formulations according tothe present invention are useful for treating and eliminating bacterialinfestations anywhere, including upper respiratory infections, topicaland systemic infections, vaginal infections, eye infections, earinfections, infections requiring parenteral treatment, as well as forthe elimination of bacteria on any surface, including human skin andmucous membrane, preferably the mucous membrane of the upper respiratorytract, more preferably the mucous membrane of the nasal cavity. Thecompositions and formulations according to the present invention areparticularly useful for the prophylaxis and treatment of upperrespiratory infections, skin infections, wounds, burns, vaginalinfections, eye infections, intestinal disorders and dental disorders.Specifically, the invention provides the application of the chimericpolypeptides for nasal and/or skin decolonisation of human and animals.

The compositions and formulations comprising a chimeric polypeptide ofthe present invention as an active ingredient are applied in aneffective amount when used in prophylaxis and therapy. The term“effective amount” refers to an amount of an active ingredientsufficient to achieve a desired effect without causing an undesirableside effect. In some cases, it may be necessary to achieve a balancebetween obtaining a desired effect and limiting the severity of anundesired effect. The amount of active ingredient used will varydepending upon the type of active ingredient and the intended use of thecomposition and/or formulation of the present invention.

In preferred embodiments, the present invention pertains to chimericpolypeptides of the invention as a prophylactic treatment for preventingthose subjects, preferably human subjects, who have possibly beenexposed to Staphylococcus bacteria, or as a therapeutic treatment forthose subjects, preferably human subjects, who have already become illfrom an infection with Staphylococcus bacteria. The chimericpolypeptides described herein are specific for decolonisation ofStaphylococcus bacteria, and preferably effectively and efficientlybreak down the cell wall of Staphylococcus bacteria, preferably ofmethicillin-resistant S. aureus (MRSA).

The chimeric polypeptides of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the chimeric polypeptide is combined in admixturewith a pharmaceutically acceptable carrier. Compositions, which may beused for the prophylactic and therapeutic treatment of a bacterialinfection, preferably a Staphylococcus bacteria infection, also includesa means of application (such as a carrier system or an oral deliverymode) to the mucosal lining of the oral and nasal cavity, such that theenzyme is put in the carrier system or oral delivery mode to reach themucosa lining.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients or stabilizers, which are non-toxic to the cell or thesubject being exposed thereto at the dosages and concentrationsemployed. Often the physiologically acceptable carrier is an aqueous pHbuffered solution. Examples of physiologically acceptable carriersinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween®, polyethylene glycol (PEG), and Pluronics®.

Prior to, or at the time a chimeric polypeptide of the invention is putin the carrier system or oral delivery mode, the enzyme may be in astabilizing buffer environment for maintaining a suitable pH range, suchas between about 5.0 and about 8.0, including a pH of about 5.0, 6.0,7.0, 8.0 or any pH interval of 0.05 there between, or any interval thatis a multiple of 0.05 there between, i.e., including for example pHvalues of 5.2, 6.5, 7.4, 7.5 and 8.5.

Therapeutic formulations are prepared for storage by mixing the activeingredient having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.

Any of the carriers for the chimeric polypeptides of the presentinvention may be manufactured by conventional means. However, if alcoholis used in the carrier, the enzyme should be in a micelle, liposome, ora “reverse” liposome, to prevent denaturing of the enzyme. Similarly,when the chimeric polypeptide is being placed in the carrier, and thecarrier is, or has been heated, such placement should be made after thecarrier has cooled somewhat, to avoid heat denaturation of the enzyme.The carrier preferably is sterile. One or more chimeric polypeptides maybe added to these substances in a liquid form or in a lyophilized state,whereupon it will be solubilized when it meets a liquid body.

A stabilizing buffer should allow for the optimum activity of thechimeric polypeptide. The buffer may contain a reducing reagent, such asdithiothreitol. The stabilizing buffer may also be or include a metalchelating reagent, such as ethylenediaminetetracetic acid disodium salt,or it may also contain a phosphate or citrate phosphate buffer, or anyother buffer.

Pharmaceuticals according to the present invention may includeanti-inflammatory agents, anti-viral agents, local anaesthetic agents,corticosteroids, destructive therapy agents, anti-fungals, and/oranti-androgens. Local anaesthetics include tetracaine, tetracainehydrochloride, lidocaine, lidocaine hydrochloride, dyclonine, dycloninehydrochloride, dimethisoquin hydrochloride, dibucaine, dibucainehydrochloride, butambenpicrate, and pramoxine hydrochloride. Anexemplary concentration for local anaesthetics is about 0.025% to about5% by weight of the total composition. Anaesthetics such as benzocainemay also be used at a preferred concentration of about 2% to about 25%by weight.

Corticosteroids that may be used include betamethasone dipropionate,fluocinolone actinide, betamethasone valerate, triamcinolone actinide,clobetasol propionate, desoximetasone, diflorasone diacetate,amcinonide, flurandrenolide, hydrocortisone valerate, hydrocortisonebutyrate, and desonide and are recommended at concentrations of about0.01% to 1.0% by weight. The concentrations for corticosteroids such ashydrocortisone or methylprednisolone acetate may be from about 0.2% toabout 5.0% by weight.

Destructive therapy agents such as salicylic acid or lactic acid mayalso be used. A concentration of about 2% to about 40% by weight may beused. Cantharidin may be utilized, for example, in a concentration ofabout 5% to about 30% by weight. Typical anti-fungals that may be usedin topical compositions and examples of suitable weight concentrationsinclude: oxiconazole nitrate (0.1% to 5.0%), ciclopirox olamine (0.1% to5.0%), ketoconazole (0.1% to 5.0%), miconazole nitrate (0.1% to 5.0%),and butoconazole nitrate (0.1% to 5.0%). Other topical agents may beincluded to address a variety of topical co-infections that may occur aswill be appreciated by skilled artisans.

In order to accelerate treatment of the infection, the therapeutic agentmay further include at least one complementary agent that can alsopotentiate the bactericidal activity of the lytic domain of the chimericpolypeptide of the invention. The complementary agent can beerythromycin, clarithromycin, azithromycin, roxithromycin, and othermembers of the macrolide family, penicillins, cephalosporins, and anycombinations thereof in amounts that are effective to synergisticallyenhance the therapeutic effect of the chimeric polypeptide of theinvention. Similarly, other lytic enzymes may be included in the carrierto treat other bacterial infections. Holin proteins may be included inthe therapeutic treatment.

In some embodiments, a mild surfactant in an amount effective topotentiate the therapeutic effect of the chimeric polypeptide may beused in or in combination with a therapeutic or prophylacticcomposition. Suitable mild surfactants include, inter alia, esters ofpolyoxyethylene sorbitan and fatty acids (Tween series), octylphenoxypolyethoxy ethanol (Triton X series), n-Octyl-β-D-glucopyranoside,n-Octyl-β-D-thioglucopyranoside, n-Decyl-β-D-glucopyranoside,n-Dodecyl-β-D-glucopyranoside, and biologically occurring surfactants,e.g., fatty acids, glycerides, monoglycerides, deoxycholate and estersof deoxycholate.

Therapeutic compositions comprising one or more chimeric polypeptides orvariants or fragments thereof can be administered or applied to asubject by any suitable means. Means of application of the chimericpolypeptide(s) (modified or unmodified) of the invention include, butare not limited to, direct, indirect, carrier and special means or anycombination of means. Direct application of the chimeric polypeptide maybe by nasal sprays, nasal drops, nasal ointments, nasal washes, nasalinjections, nasal packings, bronchial sprays and inhalers, or indirectlythrough use of throat lozenges, mouthwashes or gargles, or through theuse of ointments applied to the nasal nares, or any combination of theseand similar methods of application. The forms in which the chimericpolypeptide may be administered include but are not limited to powders,sprays, liquids, gels, ointments, and aerosols. It is most probable thatexposure to the bacteria will be through the nose. Preferred are sprays,liquids, gels, ointments, and aerosols. Particularly preferred areliquids, gels and ointments. Most preferred are liquids and gels.

When the chimeric polypeptide is introduced directly by use of nasalsprays, nasal drops, nasal ointments, nasal washes, nasal injections,nasal packing, bronchial sprays, oral sprays, or inhalers, the chimericpolypeptide may be in a liquid or gel environment, with the liquidacting as the carrier. A dry anhydrous version of the modified enzymemay be administered by the inhaler and bronchial spray, although aliquid form of delivery may also be used.

Specifically, provided herein are formulation recipes of the chimericpolypeptides of the invention in liquid aqueous matrices.

Specifically, provided herein are formulation recipes of the chimericpolypeptides of the invention in semi-solid matrices for topicalapplications.

As noted above, the chimeric polypeptide may also be placed in a nasalspray, wherein the spray is the carrier. The nasal spray can be a longacting or timed release spray, and can be manufactured by means wellknown in the art. An inhalant may also be used, so that the enzyme mayreach further down into the bronchial tract, including into the lungs.

Any of the carriers for the chimeric polypeptide may be manufactured byconventional means. However, it is preferred that any mouthwash orsimilar type products not contain alcohol to prevent denaturing of theenzyme, although enzymes in liposomes and in other protective modes andforms may be used in alcohol.

The dosage and route of administration used in a method of treatment (orprophylaxis) according to the present invention depends on the specificdisease/site of infection to be treated. The route of administration maybe, for example, in particular embodiments oral, topical,nasopharyngeal, parenteral, intravenous, rectal or any other route ofadministration.

The effective dosage rates or amounts of the enzyme(s) to treat theinfection will depend in part on whether the chimeric polypeptide willbe used therapeutically or prophylactically, the duration of exposure ofthe recipient to the infectious bacteria, the size and weight of theindividual, etc. The duration for use of the composition containing thechimeric polypeptide also depends on whether the use is for prophylacticpurposes, wherein the use may be hourly, daily or weekly, for a shorttime period, or whether the use will be for therapeutic purposes whereina more intensive regimen of the use of the composition may be needed,such that usage may last for hours, days or weeks, and/or on a dailybasis, or at timed intervals during the day. Any dosage form employedshould provide for a minimum number of units for a minimum amount oftime. The concentration of the active units of chimeric polypeptide thatmay provide for an effective amount or dosage of the chimericpolypeptide may be in the range of 10 units/ml to 500,000 units/ml offluid in the wet or damp environment of the nasal and oral passages, andtopically as well and possibly in the range of 10, 20, 30, 40, 50, 60,70, 80, 90, or 100 units/ml to 50,000 units/ml. Representative valuesthus include about 200 units/ml, 300 units/ml, 500 units/ml, 1,000units/ml, 2,500 units/ml, 5,000 units/ml, 10,000 units/ml, 20,000units/ml, 30,000 units/ml, and 40,000 units/ml. More specifically, timeexposure to the active enzyme units may influence the desiredconcentration of active enzyme units per ml. It should be noted thatcarriers that are classified as “long” or “slow” release carriers (suchas, for example, certain nasal sprays or lozenges) could possess orprovide a lower concentration of active (enzyme) units per ml, but overa longer period of time, whereas a “short” or “fast” release carrier(such as, for example, a gargle) could possess or provide a highconcentration of active (enzyme) units per ml, but over a shorter periodof time. The amount of active units per ml and the duration of time ofexposure depend on the nature of infection, whether treatment is to beprophylactic or therapeutic, and other variables. Thus, the number ofdosages will be dependent upon the circumstances and can range from 1 to4 times per day or more, with durations from one day to multiple weeks.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder, which is acondition or disorder caused by pathological bacteria, specificallyGram-positive pathological bacteria, more specifically staphylococci,more specifically Staphylococcus aureus, and most specifically MRSA.Those in need of treatment include those already with the disorder aswell as those prone to have the disorder or those for whom the disorderis to be prevented.

“Animal” for purposes of treatment refers to any animal classified as amammal, including domestic and farm animals, and zoo, sports, or petanimals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc.

The formulations to be used for in vivo administration are preferablysterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilisation andreconstitution.

The route of administration is in accordance with known methods. Whentreating a bacterial exposure or infection, the chimeric polypeptide maybe administered in any suitable fashion, including topicaladministration or through the oral or nasal cavity. For topicalapplication a polypeptide of the present invention may be administeredby way of a lotion or plaster. For nasopharyngeal application a chimericpolypeptide according to the present invention may be formulated insaline in order to be applied via a spray to the nose.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician.

It is anticipated that different formulations will be effective fordifferent treatment compounds and different disorders, thatadministration targeting one organ or tissue, for example, maynecessitate delivery in a manner different from that to another organ ortissue.

Compositions and formulations for treating topical infections comprisean effective amount of at least one chimeric polypeptide of the presentinvention produced according to this disclosure and a carrier fordelivering at least one chimeric polypeptide to the infected skin. Themode of application for the chimeric polypeptide includes a number ofdifferent types and combinations of carriers which include, but are notlimited to, an aqueous liquid, an alcohol base liquid, a water solublegel, a lotion, an ointment, a non-aqueous liquid base, a mineral oilbase, a blend of mineral oil and petrolatum, lanolin, liposomes, proteincarriers such as serum albumin or gelatin, powdered cellulose carmel,and combinations thereof. In one embodiment, a preferred carrier is anaqueous liquid. In another preferred embodiment, a preferred carrier isan alcohol base liquid. In a further embodiment, a preferred carrier isa water soluble gel. In a further embodiment, a preferred carrier is anon-aqueous liquid base. In a still further embodiment, a preferredcarrier is a lotion or an ointment.

A mode of delivery of the carrier containing the therapeutic agentincludes, but is not limited to a smear, spray, a time-release patch, aliquid absorbed wipe, and combinations thereof. A preferred mode ofdelivery of the carrier containing the therapeutic agent is a smear. Inone aspect, a preferred mode of delivery of the carrier containing thetherapeutic agent is a spray. In one embodiment, a preferred mode ofdelivery of the carrier containing the therapeutic agent is a liquidabsorbed wipe. In a further preferred embodiment, a preferred mode ofdelivery of the carrier containing the therapeutic agent is a liquidabsorbed wipe.

The chimeric polypeptide may be applied to a bandage either directly orin one of the other carriers. The bandages may be sold damp or dry,wherein the chimeric polypeptide is in a lyophilized form on thebandage. This method of application is most effective for the treatmentof infected skin.

Preservatives may also be used in this invention and may comprise, forexample, about 0.05% to 0.5% by weight of the total composition. The useof preservatives assures that if the product is microbiallycontaminated, the formulation will prevent or diminish microorganismgrowth. Some preservatives useful in this invention includemethylparaben, propylparaben, butylparaben, chloroxylenol, sodiumbenzoate, DMDM Hydantoin, 3-Iodo-2-Propylbutyl carbamate, potassiumsorbate, chlorhexidine digluconate, or a combination thereof.

All medical applications rely on the effect of the chimeric polypeptidesof the present invention to lyse specifically and immediately bacteria,preferably Gram-positive bacteria, more preferably pathogenicGram-positive bacteria, and still more preferably pathogenicstaphylococcal bacteria when encountered. This has an immediate impacton the health status of the treated subject by providing a reduction inpathogenic bacteria and bacterial load and simultaneously relieves theimmune system. Thus, the major task a person skilled in the art faces isto formulate the chimeric polypeptides of the present inventionaccurately for the respective disease to be treated. For this purposeusually the same galenic formulation as employed for conventionalmedicaments for these applications can be used.

EXAMPLES Example 1: Cloning of PRF115/Sequence=CHAP_(lysK)-Lysostaphin

PRF115 was cloned by “Splicing by overlap extension PCR” (SOE-PCR). Intwo separate PCR reactions, CHAP_(lysk) and Lysostaphin were amplified,generating overlapping fragments, which are combined to the full lengthconstruct in a third PCR. CHAP_(lysk) was amplified using pET14b_Lysk asa template with 5′T7promotor Oligonucleotide and a 3′Oligonucleotideannealing to the 3′-Terminus of the CHAP and containing 15 bases of the5′-Terminus of Lysostaphin. Lysostaphin was amplified usingpET14b_Lysostaphin as a template with a 5′Oligonucleotide annealing tothe 5′-Terminus of Lysostaphin and containing 15 bases of the3′-Terminus of CHAP_(lysK) and 3′T7-Terminator Oligonucleotide. In athird PCR reaction, the overlapping fragments of the first PCRs wereused as template and the full length PRF115 gene was amplified usingT7-Promotor and T7-Terminator Oligonucleotides. The resulting PCRProduct was (i) digested with NcoI and BamHI, ligated into pET14b andpQE60 respectively and transformed into E. Coli HMS174(DE3) and E. coliM15 respectively. The sequence was confirmed by fully sequencing thePRF115 gene.

ATGGCGAAAACCCAGGCGGAAATTAACAAACGTCTGGATGCGTATGCGAAAGGCACCGTGGATAGCCCGTATCGTGTGAAAAAAGCGACCAGCTATGATCCGAGCTTTGGCGTGATGGAAGCGGGTGCGATTGATGCGGATGGCTATTATCACGCGCAGTGCCAGGATCTGATTACCGATTATGTGCTGTGGCTGACCGATAACAAAGTGCGTACCTGGGGCAACGCGAAAGATCAGATCAAACAGAGCTATGGCACCGGCTTTAAAATCCATGAAAACAAACCGAGCACCGTGCCGAAAAAAGGCTGGATTGCGGTGTTTACCAGCGGCAGCTATGAACAGTGGGGCCATATTGGCATTGTGTATGATGGCGGCAACACCAGCACCTTTACCATTCTGGAACAGAACTGGAACGGCTATGCGAACAAAAAACCGACCAAACGCGTGGATAACTATTATGGCCTGACCCATTTTATTGAAATTCCGGTGAAAGCGGGCACCACCGTGAAAAAAGAAACCGCGAAAAAAAGCGCGAGCAAAACCCCGGCGCCGAAAAAAAAAGCCACCCTGAAAGTGAGCAAAAACCACATCAACTATACGatgGCGGCGACGCACGAGCATAGCGCCCAGTGGCTGAATAATTACAAAAAGGGTTACGGTTATGGCCCGTACCCGCTGGGCATCAATGGCGGCATGCACTATGGCGTAGACTTCTTTATGAACATTGGCACGCCGGTTAAAGCGATCAGTTCCGGTAAAATTGTGGAAGCGGGCTGGAGTAACTACGGTGGTGGTAACCAGATCGGCTTGATTGAAAATGATGGCGTGCACCGTCAGTGGTACATGCATCTGTCGAAATATAACGTAAAGGTGGGCGACTATGTGAAAGCGGGTCAAATTATTGGTTGGTCCGGTAGCACCGGTTATAGTACGGCGCCGCACCTGCATTTCCAGCGTATGGTGAATAGCTTTTCTAATAGTACCGCACAAGACCCGATGCCGTTTCTGAAATCCGCGGGTTATGGCAAAGCGGGCGGCACCGTGACTCCGACCCCGAACACGGGCTGGAAAACCAACAAGTACGGTACTCTTTACAAAAGCGAGAGCGCATCTTTTACGCCAAACACGGACATCATCACGCGCACCACCGGCCCATTTCGCAGCATGCCACAGAGCGGCGTCTTGAAAGCGGGCCAGACCATTCACTACGATGAAGTTATGAAACAGGACGGCCATGTGTGGGTGGGCTATACCGGCAACAGCGGCCAGCGTATTTATTTACCGGTTCGCACCTGGAATAAAAGCACCAATACCTTAGGCGTGTTATGGGGTACCATTAAGMAKTQAEINKRLDAYAKGTVDSPYRVKKATSYDPSFGVMEAGAIDADGYYHAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYGTGFKIHENKPSTVPKKGWIAVFTSGSYEQWGHIGIVYDGGNTSTFTILEQNWNGYANKKPTKRVDNYYGLTHFIEIPVKAGTTVKKETAKKSASKTPAPKKKATLKVSKNHINYTMAATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKIVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTAPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK

Example 2: Cloning of PRF119/Sequence=CHAP_(lysK)-CBD_(lysostaphin)

PRF119 was cloned by “Splicing by overlap extension PCR” (SOE-PCR). Intwo separate PCR reactions, CHAPlysk and CBDLysostaphin were amplified,generating an overlapping area on both fragments. CHAP_(lysk) wasamplified using pET14b_Lysk as a template with 5′T7-promotorOligonucleotide and a 3′Oligonucleotide annealing to the 3′-Terminus ofthe CHAP that contained 15 bases of the 5′-Terminus of the CBDlysostaphin. CBD_(lysostaphin) was amplified using pET14b_Lysostaphin asa template with a 5′Oligonucleotide annealing to the 5′-Terminus of theCBD_(lysostaphin) and containing 15 bases of the 3′-Terminus of CHAPlysKand 3′T7-Terminator Oligonucleotide. In a second PCR reaction, theoverlapping fragments of the first PCRs were used as template and thefull length PRF119 gene was amplified using T7Promotor and T7-TerminatorOligonucleotides. The resulting PCR Product was (i) digested with NcoIand BamHI, ligated into pET14b and pQE60 respectively and transformedinto E. Coli HMS174(DE3) and E. coli M15 respectively. The sequence wasconfirmed by fully sequencing the PRF119 gene.

ATGGCGAAAACCCAGGCGGAAATTAACAAACGTCTGGATGCGTATGCGAAAGGCACCGTGGATAGCCCGTATCGTGTGAAAAAAGCGACCAGCTATGATCCGAGCTTTGGCGTGATGGAAGCGGGTGCGATTGATGCGGATGGCTATTATCACGCGCAGTGCCAGGATCTGATTACCGATTATGTGCTGTGGCTGACCGATAACAAAGTGCGTACCTGGGGCAACGCGAAAGATCAGATCAAACAGAGCTATGGCACCGGCTTTAAAATCCATGAAAACAAACCGAGCACCGTGCCGAAAAAAGGCTGGATTGCGGTGTTTACCAGCGGCAGCTATGAACAGTGGGGCCATATTGGCATTGTGTATGATGGCGGCAACACCAGCACCTTTACCATTCTGGAACAGAACTGGAACGGCTATGCGAACAAAAAACCGACCAAACGCGTGGATAACTATTATGGCCTGACCCATTTTATTGAAATTCCGGTGATGTCTAATAGCACCGCGCAGGACCCGATGCCGTTCTTGAAGTCGGCGGGCTATGGCAAAGCAGGCGGCACCGTGACTCCGACCCCGAACACGGGCTGGAAAACCAACAAGTACGGTACTCTTTACAAAAGCGAGAGCGCATCTTTTACGCCAAACACGGACATCATCACGCGCACCACCGGCCCATTTCGCAGCATGCCACAGAGCGGCGTCTTGAAAGCGGGCCAGACCATTCACTACGATGAAGTTATGAAACAGGACGGCCATGTGTGGGTGGGCTATACCGGCAACAGCGGCCAGCGTATTTATTTACCGGTTCGCACCTGGAATAAAAGCACCAATACCTTAGGCGTGTTATGGG GTACCATTAAGTAAMAKTQAEINKRLDAYAKGTVDSPYRVKKATSYDPSFGVMEAGAIDADGYYHAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYGTGFKIHENKPSTVPKKGWIAVFTSGSYEQWGHIGIVYDGGNTSTFTILEQNWNGYANKKPTKRVDNYYGLTHFIEIPVMSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK

Example 3: Cloning of PRF102/Sequence=CHAP_(lytN)-CBD_(lysostaphin)

PRF102 was cloned by “Splicing by overlap extension PCR” (SOE-PCR). Intwo separate PCR reactions, CHAPlytN and CBD lysostaphin were amplified,generating an overlapping area on both fragments.

CHAP_(lytN) was amplified using pET14b_CHAP_(lytN) as a template with5′T7Promotor Oligonucleotide and an 3′Oligonucleotide annealing to the3′-Terminus of the CHAP_(lytN) that contained 15 bases of the5′-Terminus of the CBD_(lysostaphin). CBD_(lysostaphin) was amplifiedusing pET14b_Lysostaphin as a template with a 5′Oligonucleotideannealing to the 5′-Terminus of the CBD_(lysostaphin) and containing 15bases of the 3′-Terminus of CHAP_(lytN) and 3′T7TerminatorOligonucleotide. In a second PCR reaction, the overlapping fragments ofthe first PCRs were used as template and the full length PRF102 gene wasamplified using T7Promotor and T7Terminator Oligonucleotides. Theresulting PCR Product was (i) digested with NcoI and BamHI, ligated intopET14b and pQE60 respectively and transformed into E. Coli HMS174(DE3)and E. coli M15 respectively. The sequence was confirmed by fullysequencing the PRF102 gene.

ATGGCGAGTACATTAAATTATTTGAAAACATTAGAGAATAGAGGATGGGATTTCGACGGTAGTTATGGATGGCAATGTTTCGATTTAGTTAATGTATATTGGAATCATCTTTATGGTCATGGATTAAAAGGATATGGAGCTAAAGATATACCATATGCAAATAATTTTAATAGTGAAGCTAAAATTTATCACAACACACCAACTTTCAAAGCTGAACCTGGGGACTTAGTGGTTTTTAGTGGAAGATTTGGTGGAGGATATGGTCATACAGCTATTGTCTTAAATGGTGATTATGATGGAAAATTAATGAAGTTCCAAAGTTTAGATCAAAACTGGAATAATGGTGGATGGCGTAAAGCAGAGGTTGCACATAAAGTTGTTCATAATTATGAAAATGATATGATTTTTATTAGACCATTTAAAAAAGCAATGTCTAATAGCACCGCGCAGGACCCGATGCCGTTCTTGAAGTCGGCGGGCTATGGCAAAGCAGGCGGCACCGTGACTCCGACCCCGAACACGGGCTGGAAAACCAACAAGTACGGTACTCTTTACAAAAGCGAGAGCGCATCTTTTACGCCAAACACGGACATCATCACGCGCACCACCGGCCCATTTCGCAGCATGCCACAGAGCGGCGTCTTGAAAGCGGGCCAGACCATTCACTACGATGAAGTTATGAAACAGGACGGCCATGTGTGGGTGGGCTATACCGGCAACAGCGGCCAGCGTATTTATTTACCGGTTCGCACCTGGAATAAAAGCACCAATACCTTAGGCGTGTTATGGGGTACCATTAA GTAAMASTLNYLKTLENRGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNSEAKIYHNTPTFKAEPGDLVVFSGRFGGGYGHTAIVLNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIFIRPFKKAMSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVR TWNKSTNTLGVLWGTIK

Example 4: MIC Data

Determination of MIC values was performed in 96-well plates (Nunc,Nunclon Δ). Concentrations from 200 μg/ml to 0.00019 μg/ml of proteinwere mixed with 2×10⁴ cfu/well of Staphylococcus cell culture in BHImedium. The plate was incubated at 30° C. and optical density wasmeasured at 600 nm after 24 hours. MIC values correlate to the lowestprotein concentration at which no growth of bacteria was observed. Theresults are shown in Table 1.

TABLE 1 Minimal inhibitory concentration (MIC) Protein MIC value for S.aureus (μg/ml) PRF115 0.059 PRF119 0.39 PRF102 9.95 PRF133 0.1 LysK 87.5

Example 5: MBC Data

A Staphylococcus culture with an optical density of 0.1 was grown in BHImedium at 37° C. to an optical density of 0.8 (correlates to 10⁸Cfu/ml). Cells were harvested (4,500 rpm, 4° C., 10 minutes) andresuspended in the same volume of (i) sterile 20 mM Tris pH 7.5, 60 mMNaCl, 2 mM CaCl₂, 0.1% BSA. The culture was then diluted to 10⁵ Cfu/mlin sterile 20 mM Tris pH 7.5, 60 mM NaCl, 2 mM CaCl₂, 0.1% BSA. Thecells were mixed with protein concentrations from 50 μg/ml to 0.00005μg/ml and incubated at 30° C. for 1 hour. Dilutions from 10⁵ to 10²Cfu/ml were plated on LB-agar plates, incubated over night at 37° C. andcolonies were counted. MBC (99.99%) values correlate to the lowestprotein concentration at which a four log reduction of bacterial cellsare measured. The result is shown in Table 2.

TABLE 2 Minimal bactericidal concentration (MBC) Protein MBC (99.99%)value for S. aureus (μg/ml) PRF102 0.05

Example 6: Log Reduction in Mucin

A Staphylococcus culture with an optical density of 0.1 was grown in BHImedium at 37° C. to an optical density of 0.8 (correlates to 10⁸Cfu/ml). Cells were harvested (4,500 rpm, 4° C., 10 minutes) andresuspended in the same volume of (i) sterile 20 mM Tris pH 7.5, 60 mMNaCl, 2 mM CaCl₂ and (ii) sterile 20 mM Tris pH 7.5, 60 mM NaCl, 2 mMCaCl₂ plus 2% bovine Mucin. Samples of both preparations were mixed with0.1 mg/ml protein and dilutions were plated on Mannitol Salt Agar platesafter 1-10 minutes. Plates were incubated at 37° C. and colonies werecounted. The results are shown in Table 3.

TABLE 3 log reduction of S. aureus cells in mucin Protein Log reductionPRF119 5 PRF102 5 PRF133 6.5

Example 7: Stability Data

Thermal Stability was assayed by a photometric thermal kinetic assay.Protein solutions of 0.1 mg/ml in desired buffer were heated in quartzcuvettes 1° C. per minute from 20° C. to 95° C. and aggregation wasfollowed by measuring light dispersion at 360 nm. The temperature atwhich aggregation starts was determined as thermal changeover. Theresults are shown in Table 4.

TABLE 4 Thermal stability Protein Thermal Changeover PRF115 61° C.PRF119 54° C. PRF102 34° C. PRF133 55° C.

Long term stability was determined by incubating high concentrations ofprotein at −20° C., 4° C., 25° C. and 37° C. Samples were taken eitherweekly, daily or after hours and centrifuged. Residual proteinconcentration was determined and the lytic activity by following thedecrease of optical density in a photometric assay. Stability wasfollowed up to 1 year. Samples were also analyzed by SDS-PAGE to observeproteolytic stability. The results after storage for 38 days are shownin Table 4.1.

TABLE 4.1 Long-term stability Protein Residual activity after 38 days at37° C. PRF119  5% PRF133 85%

Example 8: Selectivity S. aureus vs. S. epidermidis vs. S. haemolyticus

Selectivity was measured by determining the MIC value for the differentStaphylococcus strains (see Example 4). The results are shown in Table5.

TABLE 5 Selectivity (MIC values in μg/ml) Protein S. aureus S.epidermidis S. haemolyticus PRF115 0.059 0.18 67 PRF119 0.39 3.125 n.d.PRF102 9.95 12.5 12.5

Example 9: In Vivo Experiment (Topical Application)

Samples of untreated skin flora were taken using LB-Agar andStaphylococcus-specific Mannitol Salt Agar contact plates from theforearm. Skin areals of 2×2 cm were treated with 10-20 μg protein bydispersing 100-200 μl of a hydrogel containing 0.1 mg/ml of protein(PRF-102). After 2 minutes, samples were again taken by LB and MSAcontact plates. Additional, skin areals were treated with a commondisinfectant (Sterilium, Fa. Bode) and samples were taken after 1minute. The plates were incubated at 37° C. over night. Residualcolonies found on MSA Agar after treatment with proteingel wereidentified as Staphylococcus epidermidis. The results are shown inFIG. 1. Topical application of a chimeric polypeptide of the presentinvention results in successful decolonisation of skin flora, as shownby the LB- and MSA-Agar plates reflecting the samples taken from humanskin treated with 10-20 μg protein containing Hydrogel.

Example 10: Formulation

2.5% Hydroxyethylcellulose (HEC), containing a buffer substance, e.g.25-50 mM sodium phosphate or Tris/HCl pH 5.5 or 7.5, and stabilizingingredients, e.g. 25 mM CaCl₂, 25 mM Citrate and 300 mM L-arginine. HECwas swollen in sterile buffer at low temperature until a homogenous gelhas formed. The protein was then dispersed in the readily swollen gel.Efficacy was shown for example by treatingStaphylococcus aureus on Agarplates. 10⁵ cells were plated on LB agar plates and shortly after theplate was dried, 100 μl of a hydrogel containing 0.1 mg/ml protein weredispersed on one half of the plate. The plate was then incubated at 37°C. over night. The results are shown in FIG. 2. Application of achimeric polypeptide of the present invention results in successfuldecolonisation of Staphylococcus aureus, as shown by the upper half ofthe Agar plate of FIG. 2B treated with gel containing 10 μg PRF102.

Although specific embodiments have been disclosed herein in some detail,this has been done solely for the purposes of describing variousfeatures and aspects of embodiments, and is not intended to be limitingwith respect to the scope of these embodiments. It is contemplated thatvarious substitutions, alterations, and/or modifications, including butnot limited to those implementation variations which may have beensuggested herein, may be made to the disclosed embodiments withoutdeparting from the spirit and scope of the embodiments as defined by theappended claims which follow.

Example 11: Cloning of PRF133/Sequence=CHAP_(lysK)-SsyntheticLinker-CBD_(lysostaphin)

PRF133 was cloned by “Splicing by overlap extension PCR” (SOE-PCR). Intwo separate PCR reactions, CHAR_(lysk) and CBD_(lysostaphin) wereamplified, generating an overlapping area on both fragments. CHAP_(lysk)was amplified using pET14b_LysK as a template with 5′-T7-promotorOligonucleotide and a 3′Oligonucleotide annealing to the 3′-Terminus ofthe CHAP that contained 33 bases of a synthetic linker sequence.CBD_(lysostaphin) was amplified using pET14b_Lysostaphin as a templatewith a 5′Oligonucleotide annealing 67 bases downstream from the5′-Terminus of CBD_(lysostaphin) and containing 33 bases of a syntheticlinker sequence and 3′-T7-Terminator Oligonucleotide. In a second PCRreaction, the overlapping fragments of the first PCRs were used astemplate and the full length PRF133 was amplified using T7-Promotor andT7-Terminator Oligonucleotides. The resulting PCR Product was digestedwith NcoI and BamHI, ligated into pET14b and pQE60 respectively. Thesequence was confirmed by fully sequencing the PRF133 gene.

ATGGCGAAAACCCAGGCGGAAATTAACAAACGTCTGGATGCGTATGCGAAAGGCACCGTGGATAGCCCGTATCGTGTGAAAAAAGCGACCAGCTATGATCCGAGCTTTGGCGTGATGGAAGCGGGTGCGATTGATGCGGATGGCTATTATCACGCGCAGTGCCAGGATCTGATTACCGATTATGTGCTGTGGCTGACCGATAACAAAGTGCGTACCTGGGGCAACGCGAAAGATCAGATCAAACAGAGCTATGGCACCGGCTTTAAAATCCATGAAAACAAACCGAGCACCGTGCCGAAAAAAGGCTGGATTGCGGTGTTTACCAGCGGCAGCTATGAACAGTGGGGCCATATTGGCATTGTGTATGATGGCGGCAACACCAGCACCTTTACCATTCTGGAACAGAACTGGAACGGCTATGCGAACAAAAAACCGACCAAACGCGTGGATAACTATTATGGCCTGACCCATTTTATTGAAATTCCGGTG GGCGGTAGCAAACCTGGAGGCACGAAGCCGGGTGGAAGCAAACCAGGATCG ACCGTGACTCCGACCCCGAACACGGGCTGGAAAACCAACAAGTACGGTACTCTTTACAAAAGCGAGAGCGCATCTTTTACGCCAAACACGGACATCATCACGCGCACCACCGGCCCATTTCGCAGCATGCCACAGAGCGGCGTCTTGAAAGCGGGCCAGACCATTCACTACGATGAAGTTATGAAACAGGACGGCCATGTGTGGGTGGGCTATACCGGCAACAGCGGCCAGCGTATTTATTTACCGGTTCGCACCTGGAATAAAAGCACCAATACCTTAGGCGTGTTATGGGGTACCATTAAGTAAMAKTQAEINKRLDAYAKGTVDSPYRVKKATSYDPSFGVMEAGAIDADGYYHAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYGTGFKIHENKPSTVPKKGWIAVFTSGSYEQWGHIGIVYDGGNTSTFTILEQNWNGYANKKPTKRVD NYYGLTHFIEIPVGGSKPGGTKPGGSKPGS TVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK

Example 12: pH-Dependent Activity of PRF119

A Staphylococcus aureus culture with an optical density of 0.1 was grownin BHI medium at 37° C. to an optical density of 1. Cells were harvested(4,500 rpm, 4° C., 10 minutes) and resupended in 10 mM Acetate, 10 mMTris, 10 mM Borate, 60 mM NaCl, 2 mM CaCl₂, pH ranging from 5-9. Afteradding 5 μg/ml protein, decrease of optical density per minute wasfollowed at 30° C. FIG. 3 shows the results.

REFERENCES

-   1. Borysowski J. et al., 2006, Exp. Biol. Med. 231: 366-377.-   2. UK patent application GB 2 255 561 A (Nov. 11, 1992).-   3. Nelson D. et al., 2001, PNAS 98(7):4107-4112.-   4. Rashel M. et al., 2007, J. Infect. Dis. 196(8):1237-1247.-   5. U.S. Pat. No. 5,997,862 (Dec. 7, 1999).-   6. Takác M. and U. Bläsi, 2005, Antimicrob. Agents Chemother.    49(7):2934-2940.-   7. Navarre W. et al., 1999, J. Biol. Chem. 274(22):15847-15856.-   8. Sass P. and G. Bierbaum, 2007, Appl. Environ. Microbiol.    73(1):347-352.-   9. O'Flaherty S. et al., 2005, J. Bacteriol. 187(20):7161-7164.-   10. WO 2008/001342 (Jan. 3, 2008)-   11. Becker S. et al., 2008, FEMS Micobiol. Lett. 287(2):185-191.-   12. Horgan M. et al., 2009, Appl. Environ. Microbiol. 75(3):872-874.-   13. Kumar Jaspal K., 2008, Appl. Microbiol. Biotechnol. 80:555-561.-   14. Kokai-Kun J. F. et al., 2007, J. Antimicrob. Chemother.    60(5):1051-1059.-   15. Kusuma C. et al., 2007, Antimicrob. Agents Chemother.    51(2):475-482.-   16. DeHart H. P. et al., 1995, Appl. Environ. Microbiol.    61(4):1475-1479.-   17. Ehlert K. et al., 1997, J. Bacteriol. 197(23):7573-7576.-   18. Strandén A. M. et al., 1997, J. Bacteriol. 197(1):9-16.-   19. Diaz E. et al., 1990, Proc. Natl. Acad. Sci. USA 87:8125-8129.-   20. Croux C. et al., 1993, Mol. Microbiol. 9(5):1019-1025.-   21. Donovan D. M. et al., 2006, Appl. Environ. Microbiol.    72(4):2988-2996.-   22. WO 2007/130655 A2 (Nov. 15, 2007)

SEQUENCE LISTING

SEQ ID NO: 1: amino acid sequence of the CHAP domain of lysK (163 aminoacid residues; translated sequence of the nucleotide sequence of SEQ IDNO: 5; origin: bacteriophage phiK)

MAKTQAEINKRLDAYAKGTVDSPYRVKKATSYDPSFGVMEAGAIDADGYYHAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYGTGFKIHENKPSTVPKKGWIAVFTSGSYEQWGHIGIVYDGGNTSTFTILEQNWNGYANKKPTKRVD NYYGLTHFIEIPV

SEQ ID NO: 2: amino acid sequence of the lytic domain of lysostaphin(124 amino acid residues; translated sequence of the nucleotide sequenceof SEQ ID NO: 6; origin: Staphylococcus simulans)

MAATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKIVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTAPHLHFQRMVNSF

SEQ ID NO: 3: amino acid sequence of the CHAP domain of lytN (143 aminoacid residues; translated sequence of the nucleotide sequence of SEQ IDNO: 7; origin: Staphylococcus aureus)

MASTLNYLKTLENRGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNSEAKIYHNTPTFKAEPGDLVVFSGRFGGGYGHTAIVLNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIFIRPFKKA

SEQ ID NO: 4: amino acid sequence of the CBD of lysostaphin (123 aminoacid residues; translated sequence of nucleotide sequence of SEQ ID NO:8; origin: Staphylococcus simulans)

SNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK

SEQ ID NO: 5: nucleotide sequence of the CHAP domain of lysK (489nucleotides; origin: bacteriophage phiK)

ATGGCGAAAACCCAGGCGGAAATTAACAAACGTCTGGATGCGTATGCGAAAGGCACCGTGGATAGCCCGTATCGTGTGAAAAAAGCGACCAGCTATGATCCGAGCTTTGGCGTGATGGAAGCGGGTGCGATTGATGCGGATGGCTATTATCACGCGCAGTGCCAGGATCTGATTACCGATTATGTGCTGTGGCTGACCGATAACAAAGTGCGTACCTGGGGCAACGCGAAAGATCAGATCAAACAGAGCTATGGCACCGGCTTTAAAATCCATGAAAACAAACCGAGCACCGTGCCGAAAAAAGGCTGGATTGCGGTGTTTACCAGCGGCAGCTATGAACAGTGGGGCCATATTGGCATTGTGTATGATGGCGGCAACACCAGCACCTTTACCATTCTGGAACAGAACTGGAACGGCTATGCGAACAAAAAACCGACCAAACGCGTGGATAACTATTATGGCCTGACCCATTTTATTGAAATTCCGGTG

SEQ ID NO: 6: nucleotide sequence of the lytic domain of lysostaphin(372 nucleotides; origin: Staphylococcus simulans)

ATGGCGGCGACGCACGAGCATAGCGCCCAGTGGCTGAATAATTACAAAAAGGGTTACGGTTATGGCCCGTACCCGCTGGGCATCAATGGCGGCATGCACTATGGCGTAGACTTCTTTATGAACATTGGCACGCCGGTTAAAGCGATCAGTTCCGGTAAAATTGTGGAAGCGGGCTGGAGTAACTACGGTGGTGGTAACCAGATCGGCTTGATTGAAAATGATGGCGTGCACCGTCAGTGGTACATGCATCTGTCGAAATATAACGTAAAGGTGGGCGACTATGTGAAAGCGGGTCAAATTATTGGTTGGTCCGGTAGCACCGGTTATAGTACGGCGCCGCACCTGCATTTCCAGCGTATGGTGAATAGCTTT

SEQ ID NO: 7: nucleotide sequence of the CHAP domain of lytN (429nucleotides; origin: Staphylococcus aureus)

ATGGCGAGTACATTAAATTATTTGAAAACATTAGAGAATAGAGGATGGGATTTCGACGGTAGTTATGGATGGCAATGTTTCGATTTAGTTAATGTATATTGGAATCATCTTTATGGTCATGGATTAAAAGGATATGGAGCTAAAGATATACCATATGCAAATAATTTTAATAGTGAAGCTAAAATTTATCACAACACACCAACTTTCAAAGCTGAACCTGGGGACTTAGTGGTTTTTAGTGGAAGATTTGGTGGAGGATATGGTCATACAGCTATTGTCTTAAATGGTGATTATGATGGAAAATTAATGAAGTTCCAAAGTTTAGATCAAAACTGGAATAATGGTGGATGGCGTAAAGCAGAGGTTGCACATAAAGTTGTTCATAATTATGAAAATGATATGATTTTTATTAGACCATTTAAAAAAGCA

SEQ ID NO: 8: nucleotide sequence of the CBD of lysostaphin (369nucleotides; origin: Staphylococcus simulans)

TCTAATAGCACCGCGCAGGACCCGATGCCGTTCTTGAAGTCGGCGGGCTATGGCAAAGCAGGCGGCACCGTGACTCCGACCCCGAACACGGGCTGGAAAACCAACAAGTACGGTACTCTTTACAAAAGCGAGAGCGCATCTTTTACGCCAAACACGGACATCATCACGCGCACCACCGGCCCATTTCGCAGCATGCCACAGAGCGGCGTCTTGAAAGCGGGCCAGACCATTCACTACGATGAAGTTATGAAACAGGACGGCCATGTGTGGGTGGGCTATACCGGCAACAGCGGCCAGCGTATTTATTTACCGGTTCGCACCTGGAATAAAAGCACCAATACCTTAGGCGT GTTATGGGGTACCATTAAG

SEQ ID NO: 9: amino acid sequence of clone PRF115 (447 amino acidresidues; translated sequence of nucleotide sequence of SEQ ID NO: 12;origin: bacteriophage phiK and Staphylococcus simulans)

MAKTQAEINKRLDAYAKGTVDSPYRVKKATSYDPSFGVMEAGAIDADGYYHAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYGTGFKIHENKPSTVPKKGWIAVFTSGSYEQWGHIGIVYDGGNTSTFTILEQNWNGYANKKPTKRVDNYYGLTHFIEIPVKAGTTVKKETAKKSASKTPAPKKKATLKVSKNHINYTMAATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKIVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTAPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK

SEQ ID NO: 10: amino acid sequence of clone PRF119 (287 amino acidresidues; translated sequence of nucleotide sequence of SEQ ID NO: 13;origin: bacteriophage phiK and Staphylococcus simulans)

MAKTQAEINKRLDAYAKGTVDSPYRVKKATSYDPSFGVMEAGAIDADGYYHAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYGTGFKIHENKPSTVPKKGWIAVFTSGSYEQWGHIGIVYDGGNTSTFTILEQNWNGYANKKPTKRVDNYYGLTHFIEIPVMSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK

SEQ ID NO: 11: amino acid sequence of clone PRF102 (267 amino acidresidues; translated sequence of nucleotide sequence of SEQ ID NO: 14;origin: Staphylococcus simulans and Staphylococcus aureus)

MASTLNYLKTLENRGWDFDGSYGWQCFDLVNVYWNHLYGHGLKGYGAKDIPYANNFNSEAKIYHNTPTFKAEPGDLVVFSGRFGGGYGHTAIVLNGDYDGKLMKFQSLDQNWNNGGWRKAEVAHKVVHNYENDMIFIRPFKKAMSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVR TWNKSTNTLGVLWGTIK

SEQ ID NO: 12: nucleotide sequence of clone PRF115 (1,341 nucleotides;origin: bacteriophage phiK and Staphylococcus simulans)

ATGGCGAAAACCCAGGCGGAAATTAACAAACGTCTGGATGCGTATGCGAAAGGCACCGTGGATAGCCCGTATCGTGTGAAAAAAGCGACCAGCTATGATCCGAGCTTTGGCGTGATGGAAGCGGGTGCGATTGATGCGGATGGCTATTATCACGCGCAGTGCCAGGATCTGATTACCGATTATGTGCTGTGGCTGACCGATAACAAAGTGCGTACCTGGGGCAACGCGAAAGATCAGATCAAACAGAGCTATGGCACCGGCTTTAAAATCCATGAAAACAAACCGAGCACCGTGCCGAAAAAAGGCTGGATTGCGGTGTTTACCAGCGGCAGCTATGAACAGTGGGGCCATATTGGCATTGTGTATGATGGCGGCAACACCAGCACCTTTACCATTCTGGAACAGAACTGGAACGGCTATGCGAACAAAAAACCGACCAAACGCGTGGATAACTATTATGGCCTGACCCATTTTATTGAAATTCCGGTGAAAGCGGGCACCACCGTGAAAAAAGAAACCGCGAAAAAAAGCGCGAGCAAAACCCCGGCGCCGAAAAAAAAAGCCACCCTGAAAGTGAGCAAAAACCACATCAACTATACGATGGCGGCGACGCACGAGCATAGCGCCCAGTGGCTGAATAATTACAAAAAGGGTTACGGTTATGGCCCGTACCCGCTGGGCATCAATGGCGGCATGCACTATGGCGTAGACTTCTTTATGAACATTGGCACGCCGGTTAAAGCGATCAGTTCCGGTAAAATTGTGGAAGCGGGCTGGAGTAACTACGGTGGTGGTAACCAGATCGGCTTGATTGAAAATGATGGCGTGCACCGTCAGTGGTACATGCATCTGTCGAAATATAACGTAAAGGTGGGCGACTATGTGAAAGCGGGTCAAATTATTGGTTGGTCCGGTAGCACCGGTTATAGTACGGCGCCGCACCTGCATTTCCAGCGTATGGTGAATAGCTTTTCTAATAGTACCGCACAAGACCCGATGCCGTTTCTGAAATCCGCGGGTTATGGCAAAGCGGGCGGCACCGTGACTCCGACCCCGAACACGGGCTGGAAAACCAACAAGTACGGTACTCTTTACAAAAGCGAGAGCGCATCTTTTACGCCAAACACGGACATCATCACGCGCACCACCGGCCCATTTCGCAGCATGCCACAGAGCGGCGTCTTGAAAGCGGGCCAGACCATTCACTACGATGAAGTTATGAAACAGGACGGCCATGTGTGGGTGGGCTATACCGGCAACAGCGGCCAGCGTATTTATTTACCGGTTCGCACCTGGAATAAAAGCACCAATACCTTAGGCGTGTTATGGGGTACCATTAAG

SEQ ID NO: 13: nucleotide sequence of clone PRF119 (864 nucleotides;origin: bacteriophage phiK and Staphylococcus simulans)

ATGGCGAAAACCCAGGCGGAAATTAACAAACGTCTGGATGCGTATGCGAAAGGCACCGTGGATAGCCCGTATCGTGTGAAAAAAGCGACCAGCTATGATCCGAGCTTTGGCGTGATGGAAGCGGGTGCGATTGATGCGGATGGCTATTATCACGCGCAGTGCCAGGATCTGATTACCGATTATGTGCTGTGGCTGACCGATAACAAAGTGCGTACCTGGGGCAACGCGAAAGATCAGATCAAACAGAGCTATGGCACCGGCTTTAAAATCCATGAAAACAAACCGAGCACCGTGCCGAAAAAAGGCTGGATTGCGGTGTTTACCAGCGGCAGCTATGAACAGTGGGGCCATATTGGCATTGTGTATGATGGCGGCAACACCAGCACCTTTACCATTCTGGAACAGAACTGGAACGGCTATGCGAACAAAAAACCGACCAAACGCGTGGATAACTATTATGGCCTGACCCATTTTATTGAAATTCCGGTGATGTCTAATAGCACCGCGCAGGACCCGATGCCGTTCTTGAAGTCGGCGGGCTATGGCAAAGCAGGCGGCACCGTGACTCCGACCCCGAACACGGGCTGGAAAACCAACAAGTACGGTACTCTTTACAAAAGCGAGAGCGCATCTTTTACGCCAAACACGGACATCATCACGCGCACCACCGGCCCATTTCGCAGCATGCCACAGAGCGGCGTCTTGAAAGCGGGCCAGACCATTCACTACGATGAAGTTATGAAACAGGACGGCCATGTGTGGGTGGGCTATACCGGCAACAGCGGCCAGCGTATTTATTTACCGGTTCGCACCTGGAATAAAAGCACCAATACCTTAGGCGTGTTATGGG GTACCATTAAGTAA

SEQ ID NO: 14: nucleotide sequence of clone PRF102 (804 nucleotides;origin: Staphylococcus simulans and Staphylococcus aureus)

ATGGCGAGTACATTAAATTATTTGAAAACATTAGAGAATAGAGGATGGGATTTCGACGGTAGTTATGGATGGCAATGTTTCGATTTAGTTAATGTATATTGGAATCATCTTTATGGTCATGGATTAAAAGGATATGGAGCTAAAGATATACCATATGCAAATAATTTTAATAGTGAAGCTAAAATTTATCACAACACACCAACTTTCAAAGCTGAACCTGGGGACTTAGTGGTTTTTAGTGGAAGATTTGGTGGAGGATATGGTCATACAGCTATTGTCTTAAATGGTGATTATGATGGAAAATTAATGAAGTTCCAAAGTTTAGATCAAAACTGGAATAATGGTGGATGGCGTAAAGCAGAGGTTGCACATAAAGTTGTTCATAATTATGAAAATGATATGATTTTTATTAGACCATTTAAAAAAGCAATGTCTAATAGCACCGCGCAGGACCCGATGCCGTTCTTGAAGTCGGCGGGCTATGGCAAAGCAGGCGGCACCGTGACTCCGACCCCGAACACGGGCTGGAAAACCAACAAGTACGGTACTCTTTACAAAAGCGAGAGCGCATCTTTTACGCCAAACACGGACATCATCACGCGCACCACCGGCCCATTTCGCAGCATGCCACAGAGCGGCGTCTTGAAAGCGGGCCAGACCATTCACTACGATGAAGTTATGAAACAGGACGGCCATGTGTGGGTGGGCTATACCGGCAACAGCGGCCAGCGTATTTATTTACCGGTTCGCACCTGGAATAAAAGCACCAATACCTTAGGCGTGTTATGGGGTACCATTAA GTAA

SEQ ID NO: 15: amino acid sequence of clone PRF133 (281 amino acidresidues; translated sequence of nucleotide sequence of SEQ ID NO: 16;origin: bacteriophage phiK and Staphylococcus simulans)

MAKTQAEINKRLDAYAKGTVDSPYRVKKATSYDPSFGVMEAGAIDADGYYHAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYGTGFKIHENKPSTVPKKGWIAVFTSGSYEQWGHIGIVYDGGNTSTFTILEQNWNGYANKKPTKRVDNYYGLTHFIEIPVGGSKPGGTKPGGSKPGSTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK

SEQ ID NO: 16: nucleotide sequence of clone PRF133 (846 nucleotides;origin: bacteriophage phiK and Staphylococcus simulans)

ATGGCGAAAACCCAGGCGGAAATTAACAAACGTCTGGATGCGTATGCGAAAGGCACCGTGGATAGCCCGTATCGTGTGAAAAAAGCGACCAGCTATGATCCGAGCTTTGGCGTGATGGAAGCGGGTGCGATTGATGCGGATGGCTATTATCACGCGCAGTGCCAGGATCTGATTACCGATTATGTGCTGTGGCTGACCGATAACAAAGTGCGTACCTGGGGCAACGCGAAAGATCAGATCAAACAGAGCTATGGCACCGGCTTTAAAATCCATGAAAACAAACCGAGCACCGTGCCGAAAAAAGGCTGGATTGCGGTGTTTACCAGCGGCAGCTATGAACAGTGGGGCCATATTGGCATTGTGTATGATGGCGGCAACACCAGCACCTTTACCATTCTGGAACAGAACTGGAACGGCTATGCGAACAAAAAACCGACCAAACGCGTGGATAACTATTATGGCCTGACCCATTTTATTGAAATTCCGGTGGGCGGTAGCAAACCTGGAGGCACGAAGCCGGGTGGAAGCAAACCAGGATCGACCGTGACTCCGACCCCGAACACGGGCTGGAAAACCAACAAGTACGGTACTCTTTACAAAAGCGAGAGCGCATCTTTTACGCCAAACACGGACATCATCACGCGCACCACCGGCCCATTTCGCAGCATGCCACAGAGCGGCGTCTTGAAAGCGGGCCAGACCATTCACTACGATGAAGTTATGAAACAGGACGGCCATGTGTGGGTGGGCTATACCGGCAACAGCGGCCAGCGTATTTATTTACCGGTTCGCACCTGGAATAAAAGCACCAATACCTTAGGCGTGTTATGGGGTACCATTAAGTAA

The invention claimed is:
 1. A chimeric polypeptide comprising a firstportion and a second portion joined by a linker, wherein (a) said firstportion comprises the amino acid sequence of a bacteriocin cell bindingdomain (CBD), wherein the CBD has greater than 90% amino acid sequenceidentity with the CBD of the chimeric polypeptide of SEQ ID NO: 11,which CBD extends from amino acid residues 145 to 267 of SEQ ID NO: 11;and (b) said second portion comprises an amino acid sequence of at leastone enzymatic active domain (EAD), wherein the EAD comprises the lyticdomain of a bacteriophage endolysin, and has greater than 90% amino acidsequence identity with the EAD of the chimeric polypeptide of SEQ ID NO:11, which EAD extends from amino acid residues 1 to 143 of SEQ ID NO:11, wherein the chimeric polypeptide has bacterial cell wall lyticactivity.
 2. The chimeric polypeptide of claim 1, wherein the linkercomprises at least one peptide bond.
 3. A nucleic acid molecule encodingthe chimeric polypeptide of claim
 1. 4. A composition comprising thechimeric polypeptide of claim
 1. 5. A topical formulation comprising thechimeric polypeptide of claim
 1. 6. The topical formulation of claim 5,which is in the form of a bioadhesive, a medicated plaster, or a skinpatch.
 7. A method of performing prophylaxis or therapy in a mammalcomprising administering to the mammal in need thereof the chimericpolypeptide of claim
 1. 8. A method of treating or preventing abacterial disease, a bacterial infection or bacterial colonization in amammal comprising administering to the mammal in need thereof thechimeric polypeptide of claim
 1. 9. The method of claim 8, wherein thechimeric polypeptide is formulated in a pharmaceutical composition. 10.The method of claim 8, wherein the polypeptide a) decreases theoccurrence or severity of a local or systemic bacterial disease orbacterial infection, or b) prevents, reduces, or eliminates bacterialcolonization.
 11. The method of claim 8, wherein the bacterial disease,bacterial infection or bacterial colonization are caused bygram-positive bacteria.
 12. The method of claim 11, wherein thegram-positive bacteria is selected from the group consisting ofStaphylococcus, Staphylococcus aureus, and methicillin-resistantStaphylococcus aureus (MRSA).
 13. The method of claim 8, wherein thebacterial disease, bacterial infection or bacterial colonization is abacterial disease, a bacterial infection or bacterial colonization ofthe skin or a mucous membrane, wherein the mucous membrane is of theupper respiratory tract, or of the nasal cavity.
 14. The chimericpolypeptide of claim 1, further comprising a pharmaceutically acceptablecarrier.
 15. The chimeric polypeptide of claim 14, wherein the carrieris aqueous, and is selected from the group consisting of a cream, a gel,a lotion, and a paste.
 16. The chimeric polypeptide of claim 1,comprising the CBD and the EAD of the chimeric polypeptide of SEQ ID NO:11, wherein the CBD extends from amino acid residues 145 to 267 of SEQID NO: 11, and the EAD extends from amino acid residues 1 to 143 of SEQID NO:
 11. 17. The chimeric polypeptide of claim 1, comprising the aminoacid sequence of the chimeric polypeptide of SEQ ID NO: 11.